Keratinocyte growth factor-2

ABSTRACT

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is a Keratinocyte Growth Factor, sometimes hereinafter referred to as “KGF-2” also formerly known as Fibroblast Growth Factor 12 (FGF-12). This invention further relates to the therapeutic use of KGF-2 to promote or accelerate wound healing. This invention also relates to novel mutant forms of KGF-2 that show enhanced activity, increased stability, higher yield or better solubility.

[0001] The present application claims benefit of the filing dates ofprovisional applications 60/055,561, filed Aug. 13, 1997 and 60/039,045,filed Feb. 28, 1997, each of which is herein incorporated by reference;the present application is also a continuation-in-part of 08/910,875,filed Aug. 13, 1997, which is herein incorporated by reference; said08/910,875 claims benefit of the filing date of provisional application60/023,852, filed Aug. 13, 1996, which is herein incorporated byreference; the present application is also a continuation-in-part of08/862,432, filed May 23, 1997, which is herein incorporated byreference; said 08/862,432 is a divisional of 08/461,195, filed Jun. 5,1995, which is herein incorporated by reference; said 08/461,195 is acontinuation-in-part of PCT/US95/01790, filed Feb. 14, 1995, which isherein incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention is a Keratinocyte Growth Factor, sometimeshereinafter referred to as “KGF-2” also formerly known as FibroblastGrowth Factor 12 (FGF-12). This invention further relates to thetherapeutic use of KGF-2 to promote or accelerate wound healing. Thisinvention also relates to novel mutant forms of KGF-2 that show enhancedactivity, increased stability, higher yield or better solubility. Inaddition, this invention relates to a method of purifying the KGF-2polypeptide.

BACKGROUND OF THE INVENTION

[0003] The fibroblast growth factor family has emerged as a large familyof growth factors involved in soft-tissue growth and regeneratior. Itpresently includes several members that share a varying degree ofhomology it the protein level, and that, with one exception, appear tohave a similar broad mitogenic spectrum, i.e., they promote theproliferation of a variety of cells of mesodermal and neuroectodermalorigin and/or promote angiogenesis.

[0004] The pattern of expression of the different members of the familyis very different, ranging from extremely restricted expressions of somestages of development, to rather ubiquitous expression in a variety oftissues and organs. All the members appear to bind heparin and heparinsulfate proteoglycans and glycosaminoglycans and strongly concentrate inthe extracellular matrix. KGF was originally identified as a member ofthe FGF family by sequence homology or factor purification and cloning.Keratinocyte growth factor (KGF) was isolated as a mitogen for acultured murine keratinocyte line (Rubin, J. S. et al., Proc. Natl.Acad. Sci. USA 86:802-806 (1989)). Unlike the other members of the FGFfamily, it has little activity on mesenchyme-derived cells butstimulates the growth of epithelial cells. The Keratinocyte growthfactor gene encodes a 194-amino acid polypeptide (Finch, P. W. et al.,Science 245:752-755 (1989)). The N-terminal 64 amino acids are unique,but the remainder of the protein has about 30% homology to bFGF. KGF isthe most divergent member of the FGF family. The molecule has ahydrophobic signal sequence and is efficiently secreted.Post-translational modifications include cleavage of the signal sequenceand N-linked glycosylation at one site, resulting in a protein of 28kDa. Keratinocyte growth factor is produced by fibroblast derived fromskin and fetal lung (Rubin et al. (1989)). The Keratinocyte growthfactor mRNA was found to be expressed in adult kidney, colon and ilium,but not in brain or lung (Finch, P. W. et al. Science 245:752-755(1989)). KGF displays the conserved regions within the FGF proteinfamily. KGF binds to the FGF-2 receptor with high affinity.

[0005] Impaired wound healing is a significant source of morbidity andmay result in such complications as dehiscence, anastomotic breakdownand, non-healing wounds. In the normal individual, wound healing, isachieved uncomplicated. In contrast, impaired healing is associated withseveral conditions such as diabetes, infection, immunosuppression,obesity and malnutrition (Cruse, P. J. and Foord, R., Arch. Surg.107:206 (1973); Schrock, T. R. et al., Ann. Surg. 177:513 (1973); Poole,G. U., Jr., Surgery 97:631 (1985); Irvin, G. L. et al., Am. Surg. 51:418(1985)).

[0006] Wound repair is the result of complex interactions and biologicprocesses. Three phases have been described in normal wound healing:acute inflammatory phase, extracellular matrix and collagen synthesis,and remodeling (Peacock, E. E., Jr., Wound Repair, 2nd edition, W BSaunders, Philadelphia (1984)). The process involves the interaction ofkeratinocytes, fibroblasts arid inflammatory cells at the wound site.

[0007] Tissue regeneration appears to be controlled by specific peptidefactors which regulate the migration and proliferation of cells involvedin the repair process (Barrett, T. B. et al., Proc. Natl. Acad. Sci. USA81:6772-6774 (1985); Collins, T. et al., Nature 316:748-750 (1985)).Thus, growth factors may be promising therapeutics in the treatment ofwounds, burns and other skin disorders (Rifkin, D. B. and Moscatelli, J.Cell. Biol. 109:1-6 (1989); Spori, M. B. et al., J. Cell. Biol.105:1039-1045 (1987); Pierce, G. F. et al., J. Cell. Biochem. 45;319-326(1991)). The sequence of the healing process is initiated during anacute inflammatory phase with the deposition of provisional tissue. Thisis followed by re-epithelialization, collagen synthesis and deposition,fibroblast proliferation, and neovascularization, all of whichultimately define the remodeling phase (Clark, R. A. F., J. Am. Acad.Dermatol. 13:701 (1985)). These events are influenced by growth factorsand cytokines secreted by inflammatory cells or by the cells localizedat the edges of the wound (Assoian, R. K. et al., Nature (Lond.) 309:804(1984); Nemeth, G. G. et al., “Growth Factors and Their Role in Woundand Fracture Healing,” Growth Factors and Other Aspects of Wound Healingin Biological and Clinical Implications, New York (1988), pp. 1-17.

[0008] Several polypeptide growth factors have been identified as beinginvolved in wound healing, including keratinocyte growth factor (KGF)(Antioniades, H. et al., Proc. Natl. Acad. Sci. USA 88:565 (1991)),platelet derived growth factor (PDGF)(Antioniades, H. et al., Proc.Natl. Acad Sci. USA 88:565 (1991); Staiano-Coico, L. et al., Jour. Exp.Med. 178:865-878 (1993)), basic fibroblast growth factor (bFGF) (Golden,M. A. et al., J. Clin. Invest. 87:406 (1991)), acidic fibroblast growthfactor (aFGF) (Mellin, T. N. et al., J. Invest. Dermatol. 104:850-855(1995)), epidermal growth factor (EGF) (Whitby, D. J. and Ferguson, W.J., Dev. Biol. 147:207 (1991)), transforming growth factor-α (TGF-α)(Gartner, M. H. et al., Surg. Forum 42:643 (1991); Todd, R. et al., Am.J. Pathol. 138;1307 (1991)), transforming growth factor-β (TGF-β) (Wong,D. T. W. et al., Am. J. Pathol. 143:622 (1987)), neu differentiationfactor (rNDF) (Danilenko, D. M. et al., J. Clin. Invest. 95;842-851(1995)), insulin-like growth factor I (IGF-1), and insulin-like growthfactor II (IGF-II) (Cromack, D. T. et al, J. Surg. Res. 42:622 (1987)).

[0009] It has been reported that rKGF-1 in the skin stimulates epidermalkeratinocytes, keratinocytes within hair follicles and sebaceous; glands(Pierce, G. F. et al., J. Exp. Med. 179:831-840 (1994)).

SUMMARY OF THE INVENTION

[0010] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the keratinocyte growth factor(KGF-2) having the amino acid sequence is shown in FIG. 1 [SEQ ID NO: 2]or the amino acid sequence encoded by the cDNA clone deposited as ATCCDeposit Number 75977 on Dec. 16, 1994. The nucleotide sequencedetermined by sequencing the deposited KGF-2 clone, which is shown inFIG. 1 [SEQ ID NO: 1], contains an open reading frame encoding apolypeptide of 208 amino acid residues, including an initiation codon atpositions 1-3, with a predicted leader sequence of about 35 or 36 aminoacid residues, and a deduced molecular weight of about 23.4 kDa. Theamino acid sequence of the mature KGF-2 is shown in FIG. 1, amino acidresidues about 36 or 37 to 208 [SEQ ID NO: 2].

[0011] The polypeptide of the present invention has been putativelyidentified as a member of the FGF family, more particularly thepolypeptide has been putatively identified as KGF-2 as a result of aminoacid sequence homology with other members of the FGF family.

[0012] In accordance with one aspect of the present invention, there areprovided novel mature polypeptides which are KGF-2 as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof. The polypeptides of thepresent invention are of human origin.

[0013] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding human KGF-2,including mRNAs, DNAs, cDNAs, genomic DNA, as well as antisense analogsthereof, and biologically active and diagnostically or therapeuticallyuseful fragments thereof.

[0014] In accordance with another aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques through the use of recombinant vectors, such as cloning andexpression plasmids useful as reagents in the recombinant production ofKGF-2 proteins, as well as recombinant prokaryotic and/or eukaryotichost cells comprising a human KGF-2 nucleic acid sequence.

[0015] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptide, orpolynucleotide encoding such polypeptide for therapeutic purposes, forexample, to stimulate epithelial cell proliferation and basalkeratinocytes for the purpose of wound healing, and to stimulate hairfollicle production and healing of dermal wounds. KGF-2 may beclinically useful in stimulating wound healing including surgicalwounds, excisional wounds, deep wounds involving damage of the dermisand epidermis, eye tissue wounds, dental tissue wounds, oral cavitywounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers,venous stasis ulcers, burns resulting from heat exposure or chemicals,and other abnormal wound healing conditions such as uremia,malnutrition, vitamin deficiencies and complications associted withsystemic treatment with steroids, radiation therapy and antineoplasticdrugs and antimetabolites. KGF-2 can be used to promote dermalreestablishment subsequent to dermal loss

[0016] KGF-2 can be used to increase the adherence of skin grafts to awound bed and to stimulate re-epithelialization from the wound bed. Thefollowing are types of grafts that KGF-2 could be used to increaseadherence to a wound bed: autografts, artificial skin, allografts,autodermic graft, autoepdermic grafts, avacular grafts, Blair-Browngrafts, bone graft, brephoplastic grafts, cutis graft, delayed graft,dermic graft, epidermic graft, fascia graft, full thickness graift,heterologous graft, xenograft, homologous graft, hyperplastic graft,lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft,omenpal graft, patch graft, pedicle graft, penetrating graft, split skingraft, thick split graft. KGF-2 can be used to promote skin strength andto improve the appearance of aged skin.

[0017] It is believed that KGF-2 will also produce changes in hepatocyteproliferation, and epithelial cell proliferation in the lung, breast,pancreas, stomach, small intesting, and large intestine. KGF-2 canpromote proliferation of epithelial cells such as sebocytes, hairfollicles, hepatocytes, type II pneumocytes, mucin-producing gobletcells, and other epithelial cells and their progenitors contained withinthe skin, lung, liver, and gastrointestinal tract. KGF-2 can promoteproliferation of endothelial cells, keratinocytes, and basalkeratinocytes.

[0018] KGF-2 can also be used to reduce the side effects of gut toxicitythat result from radiation, chemotherapy treatments or viral infections.KGF-2 may have a cytoprotective effect on the small intestine mucosa.KGF-2 may also stimulate healing of mucositis (mouth ulcers) that resultfrom chemotherapy and viral infections.

[0019] KGF-2 can further be used in full regeneration of skin in fulland partial thickness skin defects, including bums, (i.e., repopulationof hair follicles, sweat glands, and sebaceous glands), treatment ofother skin defects such as psoriasis. KGF-2 can be used to treatepidermolysis bullosa, a defect in adherence of the epidermis to theunderlying dermis which results in frequent, open and painful blistersby accelerating reepithelialization of these lesions. KGF-2 can also beused to treat gastric and doudenal ulcers and help heal by scarformation of the mucosal lining and regeneration of glandular mucosa andduodenal mucosal lining more rapidly. Inflamamatory bowel diseases, suchas Crohn's disease and ulcerative colitis, are diseases which result indestruction of the mucosal surface of the small or large intestine,respectively. Thus, KGF-2 could be used to promote the resurfacing ofthe mucosal surface to aid more rapid healing and to prevent progressionof inflammatory bowel disease. KGF-2 treatment is expected to have asignificant effect on the production of mucus throughout thegastrointestinal tract and could be used to protect the intestinalmucosa from injurious substances that are ingested or following surgery.KGF-2 can be used to treat diseases associate with the under expressionof KGF-2.

[0020] Moreover, KGF-2 can be used to prevent and heal damage to thelungs due to various pathological states. A growth factor such as KGF-2which could stimulate proliferation and differentiation and promote therepair of alveoli and brochiolar epithelium to prevent or treat acute orchronic lung damage. For example, emphysema, which results in theprogressive loss of aveoli, and inhalation injuries, i.e., resultingfrom smoke inhalation and burns, that cause necrosis of the bronchiolarepithelium and alveoli could be effectively treated KGF-2. Also, KGF-2could be used to stimulate the proliferation of and differentiation oftype II pneumocytes, which may help treat or prevent disease such ashyaline membrane diseases, such as infant respiratory distress syndromeand bronchopulmonary displasia, in premature infants.

[0021] KGF-2 could stimulate the proliferation and differentiation ofhepatocytes and, thus, could be used to alleviate or treat liverdiseases and pathologies such as fulminant liver failure caused bycirrhosis, liver damage caused by viral hepatitis and toxic substances(i.e., acetaminophen, carbon tetraholoride and other hepatotoxins knownin the art).

[0022] In addition, KGF-2 could be used treat or prevent the onset ofdiabetes mellitus. In patients with newly diagnosed Types I and IIdiabetes, where some islet cell function remains, KGF-2 could be used tomaintain the islet function so as to alleviate, delay or preventpermenant manifestation of the disease. Also, KGF-2 could be used as anauxiliary in islet cell transplantation to improve or promote islet cellfunction.

[0023] In accordance with yet a further aspect of the present invention,there are provided antibodies against such polypeptides.

[0024] In accordance with another aspect of the present invention, thereare provided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to human KGF-2 sequences.

[0025] In accordance with a further aspect of the present invention,there are provided mimetic peptides of KGF-2 which can be used astherapeutic peptides. Mimetic KGF-2 peptides are short peptides whichmimic the biological activity of the KGF-2 protein by binding to andactivating the cognate receptors of KGF-2. Mimetic KGF-2 peptides canalso bind to and inhibit the cognate receptors of KGF-2.

[0026] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides, which may be usedlo inhibit the action of such polypeptides, for example, to reducescarring during the wound healing process and to prevent and/or treattumor proliferation, diabetic retinopathy, rheumatoid arthritis,oesteoarthritis and tumor growth. KGF-2 antagonists can also be used totreat diseases associate with the over expression of KGF-2.

[0027] In accordance with yet another aspect of the present invention,there are provided diagnostic assays for detecting diseases orsusceptibility to diseases related to mutations in KGF-2 nucleic acidsequences or over-expression of the polypeptides encoded by suchsequences.

[0028] In accordance with another aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

[0029] Thus, one aspect of the invention provides an isolated nucleicacid molecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the KGF-2 polypeptide having the complete amino acid sequencein FIG. 1 [SEQ ID NO: 2]; (b) a nucleotide sequence encoding the matureKGF-2 polypeptide having the amino acid sequence at positions 36 or 37to 208 in FIG. 1 [SEQ ID NO: 2]; (c) a nucleotide sequence encoding theKGF-2 polypeptide having the complete amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 75977; (d) a nucleotidesequence encoding the mature KGF-2 polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No.75977;and (e) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c) or (d) above.

[0030] Further embodiments of the invention include isolated nucleicacid molecules that comprise a polynucleotide having a nucleotidesequence at least 90% identical, and more preferably at least 95%, 97%,98% or 99% identical, to any of the nucleotide sequences in (a), (b),(c), (d) or (e), above, or a polynucleotide which hybridizes understringent hybridization conditions to a polynucleotide in (a), (b), (c),(d) or (e), above. This polynucleotide which hybridizes does nothybridize under stringent hybridization conditions to a polynucleotidehaving a nucleotide sequence consisting of only A residues or of only Tresidues. An additional nucleic acid embodiment of the invention relatesto an isolated nucleic acid molecule comprising a polynucleotide whichencodes the amino acid sequence of an epitope-bearing portion of a KGF-2having an amino acid sequence in (a), (b), (c) or (d), above.

[0031] The invention further provides an isolated KGF-2 polypeptidehaving amino acid sequence selected from the group consisting of: (a)the amino acid sequence of the KGF-2 polypeptide having the complete 208amino acid sequence, including the leader sequence shown in FIG. 1 [SEQID NO: 2]; (b) the amino acid sequence of the mature KGF-2 polypeptide(without the leader) having the amino acid sequence at positions 36 or37 to 208 in FIG. 1 [SEQ ID NO: 2]; (c) the amino acid sequence of theKGF-2 polypeptide having the complete amino acid sequence, including theleader, encoded by the cDNA clone contained in ATCC Deposit No.75977;and (d) the amino acid sequence of the mature KGF-2 polypeptide havingthe amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 75977. The polypeptides of the present invention alsoinclude polypeptides having an amino acid sequence with at least 90%similarity, and more preferably at least 95% similarity to thosedescribed in (a), (b), (c) or (d) above, as well as polypeptides havingan amino acid sequence at least 80% identical, more preferably at least90% identical, and still more preferably 95%, 97%, 98% or 99% identicalto those above.

[0032] An additional aspect of the invention relates to a peptide orpolypeptide which has the amino acid sequence of an epitope-bearingportion of a KGF-2 polypeptide having an amino acid sequence describedin (a), (b), (c) or (d), above. Peptides or polypeptides having theamino acid sequence of an epitope-bearing portion of a KGF-2 polypeptideof the invention include portions of such polypeptides with at least sixor seven, preferably at least nine, and more preferably at least about30 amino acids to about 50 amino acids, although epitope-bearingpolypeptides of any length up to and including the entire amino acidsequence of a polypeptide of the invention described above also areincluded in the invention. In another embodiment, the invention providesan isolated antibody that binds specifically to a KGF-2 polypeptidehaving an amino acid sequence described in (a), (b), (c) or (d) above.

[0033] In accordance with another aspect of the present invention, novelvariants of KGF-2 are described. These can be produced by deleting orsubstituting one or more amino acids of KGF-2. Natural mutations arecalled allelic variations. Allelic variations can be silent (no changein the encoded polypeptide) or may have altered amino acid sequence. Inorder to attempt to improve or alter the characteristics of nativeKGF-2, protein engineering may be employed. Recombinant DNA technologyknown in the art can be used to create novel polypeptides. Muteins anddeletion mutations can show, e.g., enhanced activity or increasedstability. In addition, they could be purified in higher yield and showbetter solubility at least under certain purification and storageconditions.

[0034] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

[0035] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0036] FIGS. 1A-1C illustrate the cDNA and corresponding deduced aminoacid sequence of the polypeptide of the present invention. The initial35 or 36 amino acid residues represent the putative leader sequence(underlined). The standard one letter abbreviations for amino acids areused. Sequencing inaccuracies are a common problem when attempting todetermine polynucleotide sequences. Sequencing was performed using a 373Automated DNA sequencer (Applied Biosystems, Inc.). Sequencing accuracyis predicted to be greater than 97% accurate. [SEQ ID NO: 1]

[0037] FIGS. 2A-2D are an illustration of a comparison of the amino acidsequence of the polypeptide of the present invention and otherfibroblast growth factors. [SEQ ID NOS: 13-22]

[0038] FIGS. 3A-3D show the full length mRNA and amino acid sequence forthe KGF-2 gene. [SEQ ID NOS: 23 and 24]

[0039] FIGS. 4A-4E show an analysis of the KGF-2 amino acid sequence.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index-Jameson-Wolf” graph,amino acid residues amino acid residues 41-109 in FIG. 1 [SEQ ID NO: 2]correspond to the shown highly antigenic regions of the KGF-2 protein.Hydrophobic regions (Hopp-Woods Plot) fall below the median line(negative values) while hydrophilic regions (Kyte-Doolittle Plot) arefound above the median line (positive values, e.g. amino acid residues41-109). The plot is over the entire 208 amino acid ORF.

[0040]FIG. 5 shows the evaluation of KGF-2 on wound closure in thediabetic mice. Wounds were measured immediately after wounding and everyday for 5 consecutive days and on day 8. Percent wound closure wascalculated using the following formula: [Area on day 1]-[Area on day8]/[Area on day 1]. Statisical analysis performed using an unpaired ttest (mean+/−SEM, n=5).

[0041]FIG. 6 shows the evaluation of KGF-2 on wound closure in thenon-diabetic mice. Wounds were measured immediately after wounding andevery day for 5 consecutive days and on day 8. Percent wound closure wascalculated using the following formula: [Area on day 1]-[Area on day8]/[Area on day 1]. Statisical analysis performed using an unpaired ttest (mean+/−SEM, n=5).

[0042]FIG. 7 shows a time course of wound closure in diabetic mice.Wound areas were measured immediately after wounding and every day for 5consecutive days and on day 8. Values are presented as total area (sq.mm). Statisical analysis performed using an unpaired t test (mean+/−SEM,n=5).

[0043]FIG. 8 shows a time course of wound closure in non-diabetic mice.Wound areas were measured immediately after wounding and every day for 5consecutive days and on day 8. Values are presented as total area (sq.mm). Statisical analysis performed using an unpaired t test (mean+/−SEM,n=5).

[0044]FIG. 9 shows a histopathologic evaluation on KGF-2 on the diabeticmice. Scores were given by a blind observer. Statisical analysisperformed using an unpaired t test (mean+/−SEM, n=5).

[0045]FIG. 10 shows a histopathologic evaluation on KGF-2, on thenon-diabetic mice. Scores were given by a blind observer. Statisticalanalysis performed using an unpaired t test (mean+/−SEM, n=5).

[0046]FIG. 11 shows the effect of keratinocyte growth in the diabeticmice. Scores were given by a blind observer. Statisical analysisperformed using an unpaired t test (mean+/−SEM, n=5).

[0047]FIG. 12 shows the effect of keratinocyte growth in thenon-diabetic mice. Scores were given by a blind observer based.Statisical analysis performed using an unpaired t test (mean+/−SEM,n=5).

[0048]FIG. 13 shows the effect of skin proliferation in the diabeticmice. Scores were given by a blind observer. Statisical analysisperformed using an unpaired t test (mean+/−SEM, n=5).

[0049]FIG. 14 shows the effect of skin proliferation in the non-diabeticmice. Scores were given by a blind observer. Statisical analysisperformed using an unpaired t test (mean+/−SEM, n=5).

[0050]FIG. 15 shows the DNA sequence and the protein expressed from thepie60-Cyst37 construct [SEQ ID NOS: 29 and 30]. The expressed KGF-2protein contains the sequence from Cysteine at position 37 to Serine atposition 208 with a 6X(His) tag attached to the N-terminus of theprotein.

[0051]FIG. 16 shows the effect of methyl prednisolone on wound healingin rats. Male SD adult rats (n=5) were injected on day of wounding with5 mg of methyl prednisolone. Animals received dermal punch wounds (8 mm)and were treated daily with buffer solution or KGF-2 solution in 50 μLbuffer solution for 5 consecutive days. Wounds were measured daily ondays 1-5 and on day 8 with a calibrated Jameson caliper. Valuesrepresent measurements taken on day 8. (Mean+/−SEM)

[0052]FIG. 17 shows the effect of KGF-2 on wound closure. Male SD adultrats (n=5) received dermal punch wounds (8 mm) and 5 mg ofmethyl-prednisolone on day of wounding. Animals were treated daily witha buffer solution or KGF-2 in 50 μL of buffer solution for 5 consecutivedays commencing on the day of wounding. Measurements were made daily for5 consecutive days and on day 8. Wound closure was calculated by thefollowing formula: [Area on Day 8]-[Area on Day 1]/[Area on Day 1]. Areaon day 1 was determined to be 64 sq. mm, the area made by the dermalpunch. Statistical analysis was done using an unpaired t test.(Mean+/−SEM)

[0053]FIG. 18 shows the time course of wound healing in theglucocorticoid-impaired model of wound healing. Male SD adult rats (n=5)received dermal punch wounds (8 mm) on day 1 and were treated daily for5 consecutive days with a buffer solution or a KGF-2 solution in 50 μL.Animals received 5 mg of methyl-prednisolone on day of wounding. Woundswere measured daily for five consecutive days commencing on day ofwounding and on day 8 with a calibrated Jameson caliper. Statisticalanalysis was done using an unpaired t test. (Mean +/− SEM)

[0054]FIG. 19 (A) shows the effect of KGF-2 on wound area in rat modelof wound healing without methylprednisolone at day 5 postwounding. MaleSD rats (n=5) received dermal punch wounds (8 mm) on day 1 and weretreated daily with either a buffer solution or KGF-2 in a 50 μL solutionon day of wounding and thereafter for 5 consecutive days. Wounds weremeasured daily using a calibrated Jameson caliper. Statistical analysiswas done using an unpaired t test. (Mean+/−SEM). (B) Evaluation ofPDGF-BB and KGF-2 in Male SD Rats (n=6). All rats received 8 mm dorsalwounds and methylprednisolone (MP) (17 mg/kg) to impair wound healing.Wounds were treated daily with buffer or various concentrations ofPDGF-BB and KGF-2. Wounds were measured on Days 2, 4, 6, 8, and 10 usinga calibrated Jameson caliper. Statistical analysis was performed usingan unpaired t-test. (Mean+/−SE) *Compared with buffer. **PDGF-BB 1 μg vsKGF-2/E3 μg.

[0055]FIG. 20 shows the effect of KGF-2 on wound distance in theglucocorticoid-impaired model of wound healing. Male SD adult rats (n=5)received dermal punch wounds (8 mm) and of 17 mg/kg methyl-prednisoloneon the day of wounding. Animals were treated daily with a buffersolution or KGF-2 in 50 μL of buffer solution for 5 consecutive days andon day 8. Wound distance was measured under light microscopy with acalibrated micrometer. Statistical analysis was done using an unpaired ttest. (Mean+/−SEM)

[0056]FIG. 21 (A) shows the stimulation of normal primary epidermalkeratinocyte proliferation by KGF-2. (B) shows the stimulation of normalprimary epidermal keratinocyte proliferation by KGF-2 Δ33. (C) shows thestimulation of normal primary epidermal keratinocyte proliferation byKGF-2 Δ28. Human normal primary epidermal keratinocytes were incubatedwith various concentrations of KGF-2, KGF-2 Δ33 or KGF-2 Δ28 for theedays. For all three experiments alamarBlue was then added for 16 hr andthe intensity of the red color converted from alamarBlue by the cellswas measured by the difference between O.D. 570 nm and O.D. 600 nm. Foreach of the KGF-2 proteins a positive control with complete keratinocytegrowth media (KGM), and a negative control with keratinocyte basal media(KBM) were included in the same assay plate.

[0057]FIG. 22 (A) shows the stimulation of thymidine incorporation byKGF-2 and FGF7 in Baf3 cells transfected with FGFR1 and FGFR2. Theeffects of KGF-2 (right panel) and FGF7 (left panel) on theproliferation of Baf3 cells transfected with FGFR1iiib (open circle) orFGFR2iiib/KGFR (solid Circle were examined. Y-axis represents the amountof [3H]thymidine incoroporation (cpm) into DNA of Baf3 cells. X-axisrepresents the final concentration of KGF-2 or FGF7 added to the tissueculture media. (B) shows the stimulation of thymidine incorporation byKGF-2 Δ33 in Baf3 cells transfected with FGFR2iiib (C) shows thestimulation of thymidine incorporation by KGF-2 (white bar), KGF-2 Δ33(black bar) and KGF-2 Δ28 (grey bar) in Baf3 cells transfected withFGFR2iiib.

[0058]FIG. 23 shows the DNA and protein sequence [SEQ ID NOS: 38 and 39]for the E.coli optimized full length KGF-2.

[0059]FIGS. 24A and B show the DNA and protein sequences [SEQ ID NOS:42, 43, 54, and 55] for the E.coli optimized mature KGF-2.

[0060]FIG. 25 shows the DNA and the encoded protein sequence [SEQ IDNOS: 65 and 66] for the KGF-2 deletion construct comprising amino acids36 to 208 of KGF-2.

[0061]FIG. 26 shows the DNA and the encoded protein sequence [SEQ IDNOS: 67 and 68] for the KGF-2 deletion construct comprising amino acids63 to 208 of KGF-2.

[0062]FIG. 27 shows the DNA and the encoded protein sequence [SEQ IDNOS: 69 and 70] for the KGF-2 deletion construct comprising amino acids77 to 208 of KGF-2.

[0063]FIG. 28 shows the DNA and the encoded protein sequence [SEQ IDNOS: 71 and 72] for the KGF-2 deletion construct comprising amino acids93 to 208 of KGF-2.

[0064]FIG. 29 shows the DNA and the encoded protein sequence [SEQ IDNOS: 73 and 74] for the KGF-2 deletion construct comprising amino acids104 to 208 of KGF-2.

[0065]FIG. 30 shows the DNA and the encoded protein sequence [SEQ IDNOS: 75 and 76] for the KGF-2 deletion construct comprising am:no acids123 to 208 of KGF-2.

[0066]FIG. 31 shows the DNA and the encoded protein sequence [SEQ IDNOS: 77 and 78] for the KGF-2 deletion construct comprising am no acids138 to 208 of KGF-2.

[0067]FIG. 32 shows the DNA and the encoded protein sequence [SEQ IDNOS: 79 and 80] for the KGF-2 deletion construct comprising amino acids36 to 153 of KGF-2.

[0068]FIG. 33 shows the DNA and the encoded protein sequence [SEQ IDNOS: 81 and 82] for the KGF-2 deletion construct comprising amino acids63 to 153 of KGF-2.

[0069]FIG. 34 shows the DNA sequence for the KGF-2 Cysteine-37 to Serinemutant construct [SEQ ID NO: 83].

[0070]FIG. 35 shows the DNA sequence for the KGF-2Cysteine-37/Cysteine-106 to Serine mutant construct [SEQ ID NO: 84].

[0071]FIG. 36 shows the evaluation of KGF-2 Δ33 effects on wound healingin male SD rats (n=5). Animals received 6 mm dorsal wounds and weretreated with various concentrations buffer, or KGF-2 Δ33 for 4consecutive days. Wounds were measured daily using a calibrated Jamesoncaliper. Statistical analysis was done using an unpairedt-test.(Mean+/−SE) *Compared with buffer.

[0072]FIG. 37 shows the effect of KGF-2 Δ33 on wound healing in normalrats. Male, SD, 250-300 g, rats (n=5) were given 6 mm full-thicknessdorsal wounds. Wounds were measured with a caliper and treated withvarious concentrations of KGF-2 Δ33 and buffer for four days commencing,on the day of surgery. On the final day, wounds were harvested.Statistical analysis was performed using an unpaired t-test. *Value iscompared to No Treatment Control. †Value is compared to Buffer Control.

[0073]FIG. 38 shows the effect of KGF-2 Δ33 on breaking strength inincisional wounds. Male adult SD rats (n=10) received 2.5 cm fullthickness incisional wounds on day 1 and were intraincisionally treatedpost wounding with one application of either buffer or KGF-2 (Delta 33)(1,4, and 10 μg). Animals were sacrificed on day 5 and 0.5 cm woundspecimens were excised for routine histology, and breaking strengthanalysis. Biomechanical testing was accomplished using an Instron skintensiometer with a force applied across the wound. Breaking strength wasdefined as the greatest force withheld by each wound prior to rupture.Statistical analysis was done using an tnpaired t-test. (Mean+/−SE).

[0074]FIG. 39 shows the effect of KGF-2 (Delta 33) on epidermalthickness in incisional wounds. Male adult SD rats (n=10) received 2.5cm full thickness incisional wounds on day 1 and were intracisionallytreated postwounding with one application of either buffer or KGF-2(Delta 33) (1,4, and 10 μg). Animals were sacrificed on day 5 and 0.5 cmwound specimens were excised for routine histology and breaking strengthanalysis. Epidermal thickness was determined by taking the mean of 6measurements taken around the wound site. Measurements were taken by ablind observer on Masson Trichrome stained sections under lightmicroscopy using a calibrated lens micrometer. Statistical analysis wasdone using an unpaired t-test. (Mean+/−SE).

[0075]FIG. 40 shows the effect of KGF-2 (Delta 33) on epidermalthickness after a single intradermal injection. Male adult SD rats(n=18) received 6 intradermal injections of either buffer or KGF-2 in aconcentration of 1 and 4 μg in 50 μL on day 0. Animals were sacrificed24 and 48 hours post injection. Epidermal thickness was measured fromthe granular layer to the bottom of the basal layer. Approximately 20measurements were made along the injection site and the mean thicknessquantitated. Measurements were determined using a calibrated micrometeron Masson Trichrome stained sections under light microscopy. Statisticalanalysis was done using an unpaired t-test. (Mean +/− SE).

[0076]FIG. 41 shows the effect of KGF-2 (Delta 33) on BrdU scoring. Maleadult SD rats (n=18) received 6 intradermal injections of either placeboor KGF-2 in a concentration of 1 and 4 μg in 50 μL on day 0. Animalswere sacrificed 24 and 48 hours post injection. Animals were injectedwith 5-2′-Bromo-deoxyrudine (100 mg/kg ip) two hours prior to sacrifice.Scoring was done by a blinded observer under light microscopy using thefollowing scoring system: 0-3 none to minimal BrdU labeled cells; 4-6moderate labeling; 7-10 intense labeled cells. Statistical analysis wasdone using an unpaired t-test. (Mean+/−SE).

[0077]FIG. 42 shows the anti-inflammatory effect of KGF-2 on PAF-inducedpaw edema.

[0078]FIG. 43 shows the anti-inflammatory effect of KGF-2 Δ33 onPAF-induced paw edema in Lewis rats.

[0079]FIG. 44 shows the effect of KGF-2 Δ33 on the survival of wholebody irradiated Balb/c mice. Balb/c male mice (n=5), 22.1 g wereirradiated with 519 RADS. Animals were treated with buffer or KGF-2 (1 &5 mg/kg, s.q.) 2 days prior to irradiation and daily thereafter for 7days.

[0080]FIG. 45 shows the effect of KGF-2 Δ33 on body weight of irradiatedmice. Balb/c male mice (n=5) weighing 22.1 g were injected with eitherBuffer or KGF-2 Δ33 (1, 5 mg/kg) for 2 days prior to irradiation with519 Rad/min. The animals were weighed daily and injected for 7 daysfollowing irradiation.

[0081]FIG. 46 shows the effect of KGF-2 Δ33 on the survival rate ofwhole body irradiated Balb/c mice. Balb/c male mice (n=7), 22.1 g wereirradiated with 519 RADS. Animals were treated with buffer or KGF-2 (1and 5 mg/kg, s.q.) 2 days prior to irradiation and daily thereafter for7 days.

[0082]FIG. 47 shows the effect of KGF-2 Δ33 on wound healing in aglucocorticoid-impaired rat model.

[0083]FIG. 48 shows the effect of KGF-2 Δ33 on cell proliferation asdetermined using BrdU labeling.

[0084]FIG. 49 shows the effect of KGF-2 Δ33 on the collagen contentlocalized at anastomotic surgical sites in the colons of rats.

[0085]FIG. 50 shows a schematic representation of the pHE4-5 expressionvector (SEQ ID NO: 147) and the subcloned KGF-2 cDNA coding sequence.The locations of the kanamycin resistance marker gene, the KGF-2 codingsequence, the oriC sequence, and the lacIq coding sequence areindicated.

[0086]FIG. 51 shows the nucleotide sequence of the regulatory elementsof the pHE promoter (SEQ ID NO: 148). The two lac operator sequences,the Shine-Delgarno sequence (S/D), and the terminal HindIII and NdeIrestriction sites (italicized) are indicated.

[0087]FIG. 52 shows the proliferation of bladder epithelium following ipor sc administration of KGF-2 Δ33.

[0088]FIG. 53 shows the proliferation of prostatic epithelial cellsafter systemic administration of KGF-2 Δ33.

[0089]FIG. 54 shows the effect of KGF-2 Δ33 on bladder wall ulcerationin a cyclophosphamide-induced hemorrhagic cystitis model in the rat.

[0090]FIG. 55 shows the effect of KGF-2 Δ33 on bladder wall thickness ina cyclophosphamide-induced cystitis rat model.

[0091]FIG. 56 provides an overview of the study design to determinewhether KGF-2 Δ33 induces proliferation of normal epithelia in rats whenadministered administered systemically using SC and IP routes.

[0092]FIG. 57. Normal Sprague Dawley rats were injected daily with KGF-2Δ33 (5 mg/kg; HG03411-E2) or buffer and sacrificed one day after thefinal injection. A blinded observer counted the proliferating cells inten randomly chosen fields per animals at a 10× magnification. SCadministration of KGF-2 Δ33 elicited a significant proliferation afterone day which then returned to normal by 2 days. KGF-2 Δ33 given ipstimulated proliferation from 1-3 days but only the results from days 1and 3 were statistically significant.

[0093]FIG. 58. Normal Sprague Dawley rats were injected daily with KGF-2Δ33 (5 mg/kg; HG03411-E2) or buffer and sacrificed one day after thefinal injection. A blinded observer counted the proliferating cells inten randomly chosen fields per animal at a 10× magnification. KGF-2 Δ33given ip stimulated proliferation over the entire study period while scadministration of KGF-2 Δ33 did not increase the proliferation at anytime point.

[0094]FIG. 59. Normal Sprague Dawley rats were injected daily with KGF-2Δ33 (5 mg/kg; HG03411-E2) or buffer and sacrificed one day after thefinal injection. A blinded observer counted the proliferating cells inone cross-section per animal at a 10×magnification. KGF-2 Δ33 given scelicited a significant increase in proliferation after 1, 2, and 3 daysof daily administration. When KGF-2 Δ33 was given ip, proliferation wasseen after 2 and 3 days only.

[0095]FIG. 60 demonstrates KGF-2 Δ33 induced proliferation in normal ratlung.

DETAILED DESCRIPTION

[0096] In accordance with an aspect of the present invention, there isprovided an isolated nucleic acid (polynucleotide) which encodes for thepolypeptide having the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2) or for the polypeptide encoded by the cDNA of the clone deposited asATCC Deposit No. 75977 on Dec. 16, 1994 at the American Type CultureCollection, 12301 Park Lawn Drive, Rockville, Md. 20852.

[0097] Nucleic Acid Molecules

[0098] Unless otherwise indicated, all nucleotide sequences determinedby sequencing a DNA molecule herein were determined using an automatedDNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), andall amino acid sequences of polypeptides encoded by DNA moleculesdetermined herein were predicted by translation of a DNA sequencedetermined as above. Therefore, as is known in the art for any DNAsequence determined by this automated approach, any nucleotide sequencedetermined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

[0099] Unless otherwise indicated, each “nucleotide sequence” set forthherein is presented as a sequence of deoxyribonucleotides (abbreviatedA, G, C and T). However, by “nucleotide sequence” of a nucleic acidmolecule or polynucleotide is intended, for a DNA molecule orpolynucleotide, a sequence of deoxyribonucleotides, and for an RNAmolecule or polynucleotide, the corresponding sequence ofribonucleotides (A, G, C and U), where each thymidinedeoxyribonucleotide (T) in the specified deoxyribonuclectide sequence isreplaced by the ribonucleotide uridine (U). For instance, reference toan RNA molecule having the sequence of SEQ ID NO: 1 set forth usingdeoxyribonucleotide abbreviations is intended to indicate an RNAmolecule having a sequence in which each deoxyribonucleotide A, G or Cof SEQ ID NO: 1 has been replaced by the corresponding ribonucleotide A,G or C, and each deoxyribonucleotide T has been replaced by aribonucleotide U.

[0100] By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

[0101] Isolated nucleic acid molecules of the present invention includeDNA molecules comprising an open reading frame (ORF) with an initiationcodon at positions 1-3 of the nucleotide sequence shown in FIG. 1 (SEQID NO: 1); DNA molecules comprising the coding sequence for the matureKGF-2 protein shown in FIG. 1 (last 172 or 173 amino acids) (SEQ ID NO:2); and DNA molecules which comprise a sequence substantially differentfrom those described above but which, due to the degeneracy of thegenetic code, still encode the KJF-2 protein. Of course, the geneticcode is well known in the art. Thus, it would be routine for one skilledin the art to generate the degenerate variants described above.

[0102] A polynucleotide encoding a polypeptide of the present inventionmay be obtained from a human prostate and fetal lung. A fragment of thecDNA encoding the polypeptide was initially isolated from a libraryderived from a human normal prostate. The open reading frame encodingthe full length protein was subsequently isolated from a randomly primedhuman fetal lung cDNA library. It is structurally related to the FGFfamily. It contains an open reading frame encoding a protein of 208amino acid residues of which approximately the first 35 or 36 amino acidresidues are the putative leader sequence such that the mature proteincomprises 173 or 172 amino acids. The protein exhibits the highestdegree of homology to human keratinocyte growth factor with 45% identityand 82% similarity over a 206 amino acid stretch. It is also importantthat sequences that are conserved through the FGF family are found to beconserved in the protein of the present invention.

[0103] In addition, results from nested PCR of KGF-2 cDNA from librariesshowed that there were potential alternative spliced forms of KGF-2.Specifically, using primers flanking the N-terminus of the open readingframe of KGF-2, PCR products of 0.2 kb and 0.4 kb were obtained fromvatrious cDNA libraries. A 0.2 kb size was the expected product forKGF-2 while the 0.4 kb size may result from an alternatively splicedform of KGF-2. The 0.4 kb product was observed in libraries from stomachcancer, adult testis, duodenum and pancreas.

[0104] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be doublestranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIG. 1 (SEQ ID NO: 1) or thatof the deposited clone or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same mature polypeptide as the DNA of FIG. 1(SEQ ID NO:l) or the deposited cDNA.

[0105] The polynucleotide which encodes for the predicted maturepolypeptide of FIG. 1 (SEQ ID NO. 2) or for the predicted maturepolypeptide encoded by the deposited cDNA may include: only the codingsequence for the mature polypeptide; the coding sequence for the maturepolypeptide and additional coding sequence such as a leader or secretarysequence or a proprotein sequence; the coding sequence for the maturepolypeptide (and optionally additional coding sequence) and non-codingsequence, such as intron or non-coding sequence 5′ and/or 3′ of thecoding sequence for the predicted mature polypeptide. In addition, afull length mRNA has been obtained which contains 5′ and 3′ untranslatedregions of the gene (FIG. 3 (SEQ ID NO: 23)).

[0106] As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors discussed above, as well as thevariability of cleavage sites for leaders in different known proteins,the actual KGF-2 polypeptide encoded by the deposited cDNA comprisesabout 208 amino acids, but may be anywhere in the range of 200-220 aminoacids; and the actual leader sequence of this protein is about 35 or 36amino acids, but may be anywhere in the range of about 30 to about 40amino acids.

[0107] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0108] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIG. 1 (SEQ ID NO. 2) or the polypeptide encoded by the cDNAof the deposited clone. The variant of the polynucleotide may be anaturally occurring allelic variant of the polynucleotide or anonnaturally occurring variant of the polynucleotide.

[0109] Thus, the present invention includes polynucleotides encoding thesame predicted mature polypeptide as shown in FIG. 1 (SEQ ID NO: 2) orthe same predicted mature polypeptide encoded by the cDNA of thedeposited clone as well as variants of such polynucleotides whichvariants encode for a fragment, derivative or analog of the polypeptideof FIG. 1 (SEQ ID NO: 2) or the polypeptide encoded by the cDNA of thedeposited clone. Such nucleotide variants include deletion variants,substitution variants and addition or insertion variants.

[0110] The present invention includes polynucleotides encoding mimeticpeptides of KGF-2 which can be used as therapeutic peptides. MimeticKGF-2 peptides are short peptides which mimic the biological activity ofthe KGF-2 protein by binding to and activating the cognate receptors ofKGF-2. Mimetic KGF-2 peptides can also bind to and inhibit the cognatereceptors of KGF-2. KGF-2 receptors include, but are not limited to,FGFR2iiib and FGFR1iiib. Such mimetic peptides are obtained from methodssuch as, but not limited to, phage display or combinatorial chemistry.For example the method disclosed by Wrighton et al. Science 273:458-463(1996) to generate mimetic KGF-2 peptides.

[0111] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIG. 1 (SEQ ID NO: 1) or of the coding sequence of thedeposited clone. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encode polypeptide.

[0112] The present invention also includes polynucleotides, wherein thecoding sequence for the mature polypeptide may be fused in the samereading frame to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

[0113] Thus, for example, the polynucleotide of the present inventionmay encode for a mature protein, or for a protein having a prosequenceor for a protein having both prosequence and a presequence (leadersequence).

[0114] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexahistidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilsoni, I. et al. Cell 37:767 (1984)).

[0115] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0116] Fragments of the full length gene of the present invention may beused as a hybridization probe for a cDNA library to isolate the fulllength cDNA and to isolate other cDNAS which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 30 bases and may contain, for example, 50or more bases. The probe may also be used to identify a cDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete gene including regulatory and promotorregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or cDNA to determinewhich members of the library the probe hybridizes to.

[0117] Further embodiments of the invention include isolated nucleicacid molecules comprising a polynucleotide having a nucleotide sequenceat least 90% identical, and more preferably at least 95%, 96%, 97%, 98%or 99% identical to (a) a nucleotide sequence encoding the full-lengthKGF-2 polypeptide having the complete amino acid sequence in FIG. 1 (SEQID NO: 2), including the predicted leader sequence; (b) a nucleotidesequence encoding the mature KGF-2 polypeptide (full-length polypeptidewith the leader removed) having the amino acid sequence at positionsabout 36 or 37 to 208 in FIG. 1 (SEQ ID NO: 2); (c) a nucleotidesequence encoding the full-length KGF-2 polypeptide having the completeamino acid sequence including the leader encoded by the cDNA clonecontained in ATCC Deposit No. 75977; (d) a nucleotide sequence encodingthe mature KGF-2 polypeptide having the amino acid sequence encoded bythe cDNA clone contained in ATCC Deposit No. 75977; (e) a nucleotidesequence encoding any of the KGF-2 analogs or deletion mutants describedbelow; or (f) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c),(d), or (e).

[0118] By a polynucleotide having a nucleotide sequence at least, forexample, 95% “identical” to a reference nucleotide sequence encoding aKGF-2 polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the KGF-2polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

[0119] As a practical matter, whether any particular nucleic acidmolecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) or tothe nucleotides sequence of the deposited cDNA clone can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Reseaich Park, 575 Science Drive, Madison,Wis. 53711. Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

[0120] The present application is directed to nucleic acid molecules atleast 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIG. 1 [SEQ ID NO: 1] or to the nucleic acid sequenceof the deposited cDNA, irrespective of whether they encode a polypeptidehaving KGF-2 activity. This is because even where a particular nucleicacid molecule does not encode a polypeptide having KGF-2 activity, oneof skill in the art would still know how to use the nucleic acidmolecule, for instance, as a hybridization probe or a polymerase chainreaction (PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having KGF-2 activityinclude, inter alia, (1) isolating the KGF-2 gene or allelic variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe KGF-2 gene, as described in Verma et al., Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York (1988); andNorthern Blot analysis for detecting KGF-2 mRNA expression in specifictissues.

[0121] Preferred, however, are nucleic acid molecules having sequencesat least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acidsequence shown in FIG. 1 [SEQ ID NO: 1] or to the nucleic acid sequenceof the deposited cDNA which do, in fact, encode a polypeptide havingKGF-2 protein activity. By “a polypeptide having KGF-2 activity” isintended polypeptides exhibiting activity similar, but not necessarilyidentical, to an activity of the wild-type KGF-2 protein of theinvention or an activity that is enhanced over that of the wild-typeKGF-2 protein (either the full-length protein or, preferably, the matureprotein), as measured in a particular biological assay.

[0122] Assays of KGF-2 activity are disclosed, for example, in Examples10 and 11 below. These assays can be used to measure KGF-2 activity ofpartially purified or purified native or recombinant protein.

[0123] KGF-2 stimulates the proliferation of epidermal keratinocyes butnot mesenchymal cells such as fibroblasts. Thus, “a polypeptide havingKGF-2 protein activity” includes polypeptides that exhibit the KGF-2activity, in the keratinocyte proliferation assay set forth in Example10 and will bind to the FGF receptor isoforms 1-iiib and 2-iiib (Example11). Although the degree of activity need not be identical to that ofthe KGF-2 protein, preferably, “a polypeptide having KGF-2 proteinactivity” will exhibit substantially similar activity as compared to theKGF-2 protein (i.e., the candidate polypeptide will exhibit greateractivity or not more than about tenfold less and, preferably, not morethan about twofold less activity relative to the reference KGF-2protein).

[0124] Of course, due to the degeneracy of the genetic code, one ofordinary skill in the art will immediately recognize that a large numberof the nucleic acid molecules having a sequence at least 90%, 95%, 96%,97%, 98%, or 99% identical to the nucleic acid sequence of the depositedcDNA or the nucleic acid sequence shown in FIG. 1 [SEQ ID NO: 1] willencode a polypeptide “having KGF-2 protein activity.” In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving KGF-2 protein activity. This is because the skilled artisan isfully aware of amino acid substitutions that are either less likely ornot likely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

[0125] For example, guidance concerning how to make phenotypicallysilent amino acid substitutions is provided in Bowie, J. U. et al.,“Deciphering the Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990), wherein the authorsindicate that there are two main approaches for studying the toleranceof an amino acid sequence to change. The first method relies on theprocess of evolution, in which mutations are either accepted or rejectedby natural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselections or screens to identify sequences that maintain functionality.As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described in Bowie, J. U. et al., supra, and thereferences cited therein.

[0126] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% and stillmore preferably 96%, 97%, 98%, 99% identity between the sequences. Thepresent invention particularly relates to polynucleotides whichhybridize under stringent conditions to the hereinabove-describedpolynucleotides. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences. The polynucleotides whichhybridize to the hereinabove described polynucleotides in a preferredembodiment encode polypeptides which either retain substantially thesame biological function or activity as the mature polypeptide encodedby the cDNAs of FIG. 1 (SEQ ID NO: 1) or the deposited cDNA(s).

[0127] An example of “stringent hybridization conditions” includesovernight incubation at 42° C. in a solution comprising: 50% formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1× SSC al about 65° C.

[0128] Alternatively, the polynucleotide may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO: 1, for example, for recoveryof the polynucleotide or as a diagnostic probe or as a PCR primer.

[0129] Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide (e.g., the deposited cDNA clone), for instance,a portion 50-750 nt in length, or even to the entire length of thereference polynucleotide, are also useful as probes according to thepresent invention, as are polynucleotides corresponding to most, if notall, of the nucleotide sequence of the deposited cDNA or the nucleotidesequence as shown in FIG. 1 [SEQ ID NO: 1]. By a portion of apolynucleotide of “at least 20 nt in length,” for example, is intended20 or more contiguous nucleotides from the nucleotide sequence of thereference polynucleotide (e.g., the deposited cDNA or the nucleotidesequence as shown in FIG. 1 [SEQ ID NO: 1]). As indicated, such portionsare useful diagnostically either as a probe according to conventionalDNA hybridization techniques or as primers for amplification of a targetsequence by the polymerase chain reaction (PCR), as described, forinstance, in Molecular Cloning, A Laboratory Manual, 2nd. edition,edited by Sambrook, J., Fritsch, E. F. and Maniatis, T., (1989), ColdSpring Harbor Laboratory Press, the entire disclosure of which is herebyincorporated herein by reference.

[0130] Since a KGF-2 cDNA clone has been deposited and its determinednucleotide sequence is provided in FIG. 1 [SEQ ID NO: 1], generatingpolynucleotides which hybridize to a portion of the KGF-2 cDNA moleculewould be routine to the skilled artisan. For example, restrictionendonuclease cleavage or shearing by sonication of the KGF-2 cDNA clonecould easily be used to generate DNA portions of various sizes which arepolynucleotides that hybridize to a portion of the KGF-2 cDNA molecule.Alternatively, the hybridizing polynucleotides of the present inventioncould be generated synthetically according to known techniques. Ofcourse, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of the KGF-2 cDNA shown in FIG. 1[SEQ ID NO: 1]), or to a complementary stretch of T (or U) resides,would not be included in a polynucleotide of the invention used tohybridize to a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

[0131] The invention further provides isolated nucleic acid moleculescomprising a polynucleotide encoding an epitope-bearing portion of theKGF-2 protein. In particular, isolated nucleic acid molecules areprovided encoding polypeptides comprising the following amino acidresidues in FIG. 1 (SEQ ID NO: 2), which the present inventors havedetermined are antigenic regions of the KGF-2 protein: 1. Gly41-Asn71:GQDMVSPEATNSSSSSFSSPSSAGRJIVRSYN; [SEQ ID NO:25] 2. Lys91-Ser109:KIEKNGKVSGTKKENCPYS; [SEQ ID NO:26] 3. Asn135-Tyr164:NKKGKLYGSKEFNNDCKLKERIEENGYNTY; and [SEQ ID NO 27] 4. Asn181-Ala199:NGKGAPRRGQKTRRKNTSA. [SEQ ID NO:28]

[0132] Also, there are two additonal shorter predicted antigenic areas,Gln74-Arg78 of FIG. 1 (SEQ ID NO: 2) and Gln170-Gln175 of FIG. 1 (SEQ IDNO: 2). Methods for gererating such epitope-bearing portions of KGF-2are described in detail below.

[0133] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0134] KGF-2 Polypeptides and Fragments

[0135] The present invention further relates to a polypeptide which hasthe deduced amino acid sequence of FIG. 1 (SEQ ID NO: 2) or which hasthe amino acid sequence encoded by the deposited cDNA, as well asfragments, analogs and derivatives of such polypeptide.

[0136] As one of ordinary skill would appreciate, due to thepossibilities of sequencing errors discussed above, as well as thevariability of cleavage sites for leaders in different known proteins,the actual KGF-2 polypeptide encoded by the deposited cDNA comprisesabout 208 amino acids, but may be anywhere in the range of 200-220 aminoacids; and the actual leader sequence of this protein is about 35 or 36amino acids, but may be anywhere in the range of about 30 to about 40amino acids.

[0137] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide, of FIG. 1 (SEQ ID NO: 2) or that encoded by thedeposited cDNA, means a polypeptide which retains essentially the samebiological function or activity as such polypeptide. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

[0138] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

[0139] The fragment, derivative or analog of the polypeptide of FIG. 1(SEQ ID NO: 2) or that encoded by the deposited cDNA may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretarysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0140] The terms “peptide” and “oligopeptide” are considered synonymous(as is commonly recognized) and each term can be used interchangeably asthe context requires to indicate a chain of at least to amino acidscoupled by peptidyl linkages. The word “polypeptide” is used herein forchains containing more than ten amino acid residues. All oligopeptideand polypeptide formulas or sequences herein are written from left toright and in the direction from amino terminus to carboxy terminus.

[0141] It will be recognized in the art that some amino acid sequencesof the KGF-2 polypeptide can be varied without significant effect of thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity. In general, it ispossible to replace residues which form the tertiary structure, providedthat residues performing a similar function are used. In otherinstances, the type of residue may be completely unimportant if thealteration occurs at a non-critical region of the protein.

[0142] Thus, the invention further includes variations of the KGF-2polypeptide which show substantial KGF-2 polypeptide activity or whichinclude regions of KGF-2 protein such as the protein portions discussedbelow. Such mutants include deletions, insertions, inversions, repeats,and type substitutions (for example, substituting one hydrophilicresidue for another, but not strongly hydrophilic for stronglyhydrophobic as a rule). Small changes or such “neutral” amino acidsubstitutions will generally have little effect on activity.

[0143] Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchangeof the acidic residues Asp and Glu, substitution between the amideresidues Asn and Gln, exchange of the basic residues Lys and Arg andreplacements among the aromatic residues Phe, Tyr.

[0144] As indicated in detail above, further guidance concerning whichamino acid changes are likely to be phenotypically silent (i.e., are notlikely to have a significant deleterious effect on a function) can befound in Bowie, J. U., et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990).

[0145] The present invention includes mimetic peptides of KGF-2 whichcan be used as therapeutic peptides. Mimetic KGF-2 peptides are shortpeptides which mimic the biological activity of the KGF-2 protein bybinding to and activating the cognate receptors of KGF-2. Mimetic KGF-2peptides can also bind to and inhibit the cognate receptors of KGF-2.KGF-2 receptors include, but are not limited to, FGFR2iiib andFGFR1iiib. Such mimetic peptides are obtained from methods such as, butnot limited to, phage display or combinatorial chemistry. For example,the method disclosed by Wrighton et al. Science , 273:458-463 (1996) canbe used to generate mimetic KGF-2 peptides.

[0146] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0147] The polypeptides of the present invention are preferably in anisolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontaned within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended are polypeptides thathave been purified, partially or substantially, from a recombinant hostcell or a native source.

[0148] The polypeptides of the present invention include the polypeptideof SEQ ID NO: 2 (in Particular the mature polypeptide) as well aspolypeptides which have at least 90%, 95%, 96%, 97%, 98%, 99% similarity(more preferably at least 90%, 95%, 96%, 97%, 98%, 99% identity) to thepolypeptide of SEQ ID NO: 2 and also include portions of suchpolypeptides with such portion of the polypeptide (such as the deletionmutants described below) generally containing at least 30 amino acidsand more preferably at least 50 amino acids.

[0149] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0150] By “% similarity” for two polypeptides is intended a similarityscore produced by comparing the amino acid sequences of the twopolypeptides using the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711) and the defaultsettings for determining similarity. Bestfit uses the local homologyalgorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the best segment of similarity between twosequences.

[0151] By a polypeptide having an amino acid sequence at least, forexample, 95% “identical” to a reference amino acid sequence of a KGF-2polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the KGF-2 polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0152] As a practical matter, whether any particular polypeptide is atleast 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence shown in FIG. 1 [SEQ ID NO: 2] or to the amino acidsequence encoded by deposited cDNA clone can be determinedconventionally using known computer programs such the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Reseaich Park, 575 Science Drive, Madison,Wis. 53711. When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

[0153] As described in detail below, the polypeptides of the presentinvention can be used to raise polyclonal and monoclonal antibodies,which are useful in diagnostic assays for detecting KGF-2 proteinexpression as described below or as agonists and antagonists capable ofenhancing or inhibiting KGF-2 protein function. Further, suchpolypeptides can be used in the yeast two-hybrid system to “capture”KGF-2 protein binding proteins which are also candidate agonist andantagonist according to the present invention. The yeast two hybridsystem is described in Fields and Song, Nature 340:245-246 (1989).

[0154] In another aspect, the invention provides a peptide orpolypeptide comprising an epitope-bearing portion of a polypeptide ofthe invention. The epitope of this polypeptide portion is an immunogenicor antigenic epitope of a polypeptide of the invention. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein is the immunogen. These immunogenicepitopes are believed to be confined to a few loci on the molecule. Onthe other hand, a region of a protein molecule to which an antibody canbind is defined as an “antigenic epitope.” The number of immunogenicepitopes of a protein generally is less than the number of antigenicepitopes. See, for instance, Geysen et al, Proc. Natl. Acad Sci. USA81:3998-4002 (1983).

[0155] As to the selection of peptides or polypeptides bearing anantigenic epitope (i.e., that contain a region of a protein molecule towhich an antibody can bind), it is well known in that art thatrelatively short synthetic peptides that mimic part of a proteinsequence are routinely capable of eliciting an antiserum that reactswith the partially mimicked protein. See, for instance, Sutcliffe, J.G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies thatreact with predetermined sites on proteins. Science 219:660-666.Peptides capable of eliciting protein-reactive sera are frequentlyrepresented in the primary sequence of a protein, can be characterizedby a set of simple chemical rules, and are confined neither toimmunodominant regions of intact proteins (i.e., immunogenic epitopes)nor to the amino or carboxyl terminals. Peptides that are extremelyhydrophobic and those of six or fewer residues generally are ineffectiveat inducing antibodies that bind to the mimicked protein; longer,soluble peptides, especially those containing proline residues, usuallyare effective. Sutcliffe et al., supra, at 661. For instance, 18 of 20peptides designed according to these guidelines, containing 8-39residues covering 75% of the sequence of the influenza virushemagglutinin HA1 polypeptide chain, induced antibodies that reactedwith the HA1 protein or intact virus; and 12/12 peptides from the MuLVpolymerase and 18/18 from the rabies glycoprotein induced antibodiesthat precipitated the respective proteins.

[0156] Antigenic epitope-bearing peptides and polypeptides of theinvention are therefore useful to raise antibodies, including monoclonalantibodies, that bind specifically to a polypeptide of the invention.Thus, a high proportion of hybridomas obtained by fusion of spleen cellsfrom donors immunized with an antigen epitope-bearing peptide generallysecrete antibody reactive with the native protein. Sutcliffe et al.,supra, at 663. The antibodies raised by antigenic epitope-bearingpeptides or polypeptides are useful to detect the mimicked protein, andantibodies to different peptides may be used for tracking the fate ofvarious regions of a protein precursor which undergoes posttranslational processing. The peptides and anti-peptide antibodies maybe used in a variety of qualitative or quantitative assays for themimicked protein, for instance in competition assays since it has beenshown that even short peptides (e.g., about 9 amino acids) can bind anddisplace the larger peptides in immunoprecipitation assays. See, forinstance, Wilson et al., Cell 37:767-778 (1984) at 777. The anti-peptideantibodies of the invention also are useful for purification of themimicked protein, for instance, by adsorption chromatography usingmethods well known in the art.

[0157] Antigenic epitope-bearing peptides and polypeptides of theinvention designed according to the above guidelines preferably containa sequence of at least seven, more preferably at least nine and mostpreferably between about 15 to about 30 amino acids contained within theamino acid sequence of a polypeptide of the invention. However, peptidesor polypeptides comprising a larger portion of an amino acid sequence ofa polypeptide of the invention, containing about 30, 40, 50, 60, 70, 80,90, 100, or 150 amino acids, or any length up to and including theentire amino acid sequence of a polypeptide of the invention, also areconsidered epitope-bearing peptides or polypeptides of the invention andalso are useful for inducing antibodies that react with the mimickedprotein. Preferably, the amino acid sequence of the epitope-bearingpeptide is selected to provide substantial solubility in aqueoussolvents (i.e., the sequence includes relatively hydrophilic residuesand highly hydrophobic sequences are preferably avoided); and sequencescontaining proline residues are particularly preferred.

[0158] Non-limiting examples of antigenic polypeptides or peptides thatcan be used to generate KGF-2-specific antibodies include thefollowing: 1. Gly41-Asn71: GQDMVSPEATNSSSSSFSSPSSAGRHVRSYN; [SEQ IDNO:25] 2. Lys91-Ser109: KIEKNGKVSGTKKENCPYS; [SEQ ID NO:26] 3.Asn135-Tyr164: NKKGKLYGSKEFNNDCKLKERIEENGYNTY; and [SEQ ID NO:27] 4.Asn181-Ala199: NGKGAPRRGQKTRRKNTSA. [SEQ ID NO:28]

[0159] Also, there are two additonal shorter predicted antigenic areas,Gln74-Arg78 of FIG. 1 (SEQ ID NO: 2) and Gln170-Gln175 of FIG. 1 (SEQ IDNO: 2).

[0160] The epitope-bearing peptides and polypeptides of the inventionmay be produced by any conventional means for making peptides orpolypeptides including recombinant means using nucleic acid molecules ofthe invention. For instance, a short epitope-bearing amino acid sequencemay be fused to a larger polypeptide which acts as a carrier duringrecombinant production and purification, as well as during immunizationto produce anti-peptide antibodies. Epitope-bearing peptides also may besynthesized using known methods of chemical synthesis. For instance,Houghten has described a simple, method for synthesis of large numbersof peptides, such as 10-20 mg of 248 different 13 residue peptidesrepresenting single amino acid variants of a segment of the HA1polypeptide which were prepared and characterized (by ELISA-type bindingstudies) in less than four weeks. Houghten, R. A. (1985) General methodfor the rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids. Proc. Natl. Acad Sci. USA 82:5131-5135. This “SimultaneousMultiple Peptide Synthesis (SMPS)” process is further described in U.S.Pat. No. 4,631,211 to Houghten et al. (1986). In this procedure theindividual resins for the solid-phase synthesis of various peptides arecontained in separate solvent-permeable packets, enabling the optimaluse of the many identical repetitive steps involved in solid-phasemethods. A completely manual procedure allows 500-1000 or more synthesesto be conducted simultaneously. Houghten et al., supra, at 5134.

[0161] Epitope-bearing peptides and polypeptides of the invention areused to induce antibodies according to methods well known in the art.See for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow,M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. etal., J. Gen. Virol. 66:2347-2354 (1985). Generally, animals may beimmunized with free peptide; however, anti-peptide antibody titer may beboosted by coupling of the peptide to a macromolecular carrier, such askeyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance,peptides containing cysteine may be coupled to carrier using a linkersuch as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg peptide or carrier protein and Freund's adjuvant. Severalbooster injections may be needed, for instance, at intervals of abouttwo weeks, to provide a useful titer of anti-peptide antibody which canbe detected, for example, by ELISA assay using free peptide adsorbed toa solid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

[0162] Immunogenic epitope-bearing peptides of the invention, i.e.,those parts of a protein that elicit an antibody response when the wholeprotein is the immunogen, are identified according to methods known inthe art. For instance, Geysen et al., supra, discloses a procedure forrapid concurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an enzyme-linked immunosorbent assay.Interaction of synthesized peptides with antibodies is then easilydetected without removing them from the support. In this manner apeptide bearing an immunogenic epitope of a desired protein may beidentified routinely by one of ordinary skill in the art. For instance,the immunologically important epitope in the coat protein offoot-and-mouth disease virus was located by Geysen et al. with aresolution of seven amino acids by synthesis of an overlapping set ofall 208 possible hexapeptides covering the entire 213 amino acidsequence of the protein. Then, a complete replacement set of peptides inwhich all 20 amino acids were substituted in turn at every positionwithin the epitope were synthesized, and the particular amino acidsconferring specificity for the reaction with antibody were determined.Thus, peptide analogs of the epitope-bearing peptides of the inventioncan be made routinely by this method. U.S. Pat. No. 4,708,781 to Geysen(1987) further describes this method of identifying a peptide bearing animmunogenic epitope of a desired protein.

[0163] Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describesa general method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C₁-C₇-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

[0164] As one of skill in the art will appreciate, KGF-2 polypeptides ofthe present invention and the epitope-bearing fragments thereofdescribed above can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate, purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric KGF-2 protein or proteinfragment alone (Fountoulakis et al, J Biochem 270:3958-3964 (1995)).

[0165] In accordance with the present invention, novel variants of KGF-2are also described. These can be produced by deleting or substitutingone or more amino acids of KGF-2. Natural mutations are called allelicvariations. Allelic variations can be silent (no change in the encodedpolypeptide) or may have altered amino acid sequence.

[0166] In order to attempt to improve or alter the characteristics ofnative KGF-2, protein engineering may be employed. Recombinant DNAtechnology known to those skilled in the art can be used to create novelpolypeptides. Muteins and deletions can show, e.g., enhanced activity orincreased stability. In addition, they could be purified in higher yieldand show better solubility at least under certain purification andstorage conditions. Set forth below are examples of mutations that canbe constructed.

[0167] Amino Terminal and Carboxy Terminal Deletions

[0168] Various members of the FGF family have been modified usingrecombinant DNA technology. Positively charged molecules have beensubstituted or deleted in both aFGF and bFGF that are important forheparin binding. The modified molecules resulted in reduced heparinbinding activity. Accordingly, it is known that the amount of modifiedmolecule sequestered by heparin in a patient would be reduced,increasing the potency as more FGF would reach the appropriate receptor.(EP 0 298 723).

[0169] Native KGF-2 is relatively unstable in the aqueous state and itundergoes chemical and physical degradation resulting in loss ofbiological activity during processing and storage. Native KGF-2 is alsoprone to aggregation in aqueous solution, at elevated temperatures andit becomes inactivated under acidic conditions.

[0170] In order to improve or alter one or more characteristics ofnative KGF-2, protein enginerring may be employed. Ron et al., J. Biol.Chem., 268(4): 2984-2988 (1993) reported modified KGF proteins that hadheparin binding activity even if the 3, 8, or 27 amino terminal aminoacid residues were missing. The deletion of 3 and 8 amino acids had fullactivity. More deletions of KGF have been descibed in PCT/IB95/00971.The deletion of carboxyterminal amino acids can enhance the activity ofproteins. One example is interferon gamma that shows up to ten timeshigher activity by deleting ten amino acid residues from the carboxyterminus of the protein (Döbeli et al., J. of Biotechnology 7:199-216(1988)). Thus, one aspect of the invention is to provide polypeptideanalogs of KGF-2 and nucleotide sequences encoding such analogs thatexhibit enhanced stability (e.g., when exposed to typical pH, thermalconditions or other storage conditions) relative to the native KGF-2polypeptide.

[0171] Particularly preferred KGF-2 polypeptides are shown below(numbering starts with the first amino acid in the protein (Met) (FIG. 1(SEQ ID NO: 2)): Thr (residue 36) -- Ser (residue 208) Cys (37) -- Ser(208) Gln (38) -- Ser (208) Ala (39) -- Ser (208) Leu (40) -- Ser (208)Gly (41) -- Ser (208) Gln (42) -- Ser (208) Asp (43) -- Ser (208) Met(44) -- Ser (208) Val (45) -- Ser (208) Ser (46) -- Ser (208) Pro (47)-- Ser (208) Gln (48) -- Ser (208) Ala (49) -- (Ser (208) Thr (50) --Ser (208) Asn (51) -- Ser (208) Ser (52) -- Ser (208) Ser (53) -- Ser(208) Ser (54) -- Ser (208) Ser (55) -- Ser (208) Ser (56) -- Ser (208)Phe (57) -- Ser (208) Ser (59) -- Ser (208) Ser (62) -- Ser (208) Ala(63) -- Ser (208) Gly (64) -- Ser (208) Arg (65) -- Ser (208) Val (67)-- Ser (208) Ser (69) -- Ser (208) Val (77) -- Ser (208) Arg (80) -- Ser(208) Met(1), Thr (36), or Cys (37) -- His (207) Met (1), Thr (36), orCys (37) -- Val (206) Met (1), Thr (36), or Cys (37) -- Val (205)Met(1), Thr (36), or Cys (37) -- Met (204) Met(1), Thr (36), or Cys (37)-- Pro (203) Met(1), Thr (36), or Cys(37) -- Leu (202) Met(1), Thr (36),or Cys (37) -- Phe (201) Met(1), Thr (36), or Cys (37) -- His (200)Met(1), Thr (36), or Cys (37) -- Ala (199) Met(1), Thr (36), or Cys (37)-- Ser (198) Met (1), Thr (36), or Cys (37) -- Thr (197) Met(1), Thr(36), or Cys (37) -- Asn (196) Met(1), Thr (36), or Cys (37) -- Lys(195) Met (1), Thr (36), or Cys (37) -- Arg (194) Met(1), Thr (36), orCys (37) -- Arg (193) Met(1), Thr (36), or Cys (37) -- Thr (192) Met(1),Thr (36), or Cys (37) -- Lys (191) Met(1), Thr (36), or Cys (37) -- Arg(188) Met(1), Thr (36), or Cys (37) -- Arg (187) Met(1), Thr (36), orCys (37) -- Lys (183)

[0172] Preferred embodiments include the N-terminal deletions Ala(63)-Ser (208) (KGF-2Δ28) (SEQ ID NO: 68) and Ser (69)-Ser (208)(KGF-2Δ33) (SEQ ID NO: 96). Other preferred N-terminal and C-terminaldeletion mutants are described in Examples 13 and 16 (c) of thespecification and include: Ala (39)-Ser (208) (SEQ ID NO: 116); Pro(47)-Ser (208) of FIG. 1 (SEQ ID NO: 2); Val (77)-Ser (208) (SEQ ID NO:70); Glu (93)-Ser (208) (SEQ ID NO: 72); Glu (104)-Ser (208) (SEQ ID NO:74); Val (123)-Ser (208) (SEQ ID NO: 76); and Gly (138)-Ser (208) (SEQID NO: 78). Other preferred C-terminal deletion mutants include: Met(1), Thr (36), or Cys (37)-Lys (153) of FIG. 1 (SEQ ID NO: 2).

[0173] Also included by the present invention are deletion mutantshaving amino acids deleted from both the N-terminus and the C-terminus.Such mutants include all combinations of the N-terminal deletion mutantsand C-terminal deletion mutants described above, e.g., Ala (39)-His(200) of FIG. 1 (SEQ ID NO: 2), Met (44)-Arg (193) of FIG. 1 (SEQ ID NO:2), Ala (63)-Lys (153) of FIG. 1 (SEQ ID NO: 2), Ser (69)-Lys (153) ofFIG. 1 (SEQ ID NO: 2), etc. etc. etc. . . . Those combinations can bemade using recombinant techniques known to those skilled in the art.

[0174] Thus, in one aspect, N-terminal deletion mutants are provided bythe present invention. Such mutants include those comprising the aminoacid sequence shown in FIG. 1 (SEQ ID NO: 2) except for a deletion of atleast the first 38 N-terminal amino acid residues (i.e., a deletion ofat least Met (1)-Gln (38)) but not more than the first 147 N-terminalamino acid residues of FIG. 1 (SEQ ID NO: 2). Alternatively, thedeletion will include at least the first 38 N-terminal amino acidresidues (i.e., a deletion of at least Met (1)-Gln (38)) but not morethan the first 137 N-terminal amino acid residues of FIG. 1 (SEQ ID NO:2). Alternatively, the deletion will include at least the first 46N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2). Alternatively,the deletion will include at least the first 62 N-terminal amino acidresidues but not more than the first 137 N-terminal amino acid residuesof FIG. 1 (SEQ ID NO: 2). Alternatively, the deletion will include atleast the first 68 N-terminal amino acid residues but not more than thefirst 137 N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2).Alternatively, the deletion will include at least the first 76N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2). Alternatively,the deletion will include at least the first 92 N-terminal amino acidresidues but not more than the first 137 N-terminal amino acid residuesof FIG. 1 (SEQ ID NO: 2). Alternatively, the deletion will include atleast the first 103 N-terminal amino acid residues but not more than thefirst 137 N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2).Alternatively, the deletion will include at least the first 122N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2).

[0175] In addition to the ranges of N-terminal deletion mutantsdescribed above, the present invention is also directed to allcombinations of the above described ranges, e.g., deletions of at leastthe first 62 N-terminal amino acid residues but not more than the first68 N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2); deletions ofat least the first 62 N-terminal amino acid residues but not more thanthe first 76 N-terminal amino acid residues of FIG.1 (SEQ ID NO: 2);deletions of at least the first 62 N-terminal amino acid residues butnot more than the first 92 N-terminal amino acid residues of FIG. 1 (SEQID NO: 2); deletions of at least the first 62 N-terminal amino acidresidues but not more than the first 103 N-terminal amino acid residuesof FIG. 1 (SEQ ID NO: 2); deletions of at least the first 68 N-terminalamino acid residues but not more than the first 76 N-terminal amino acidresidues of FIG. 1 (SEQ ID NO: 2); deletions of at least the first 68N-terminal amino acid residues but not more than the first 92 N-terminalamino acid residues of FIG. 1 (SEQ ID NO: 2); deletions of at least thefirst 68 N-terminal amino acid residues but not more than the first 103N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2); deletions of atleast the first 46 N-terminal amino acid :residues but not more than thefirst 62 N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2);deletions of at least the first 46 N-terminal amino acid residues butnot more than the first 68 N-terminal amino acid residues of FIG. 1 (SEQID NO: 2); deletions of at least the first 46 N-terminal amino acidresidues but not more than the first 76 N-terminal amino acid residuesof FIG. 1 (SEQ ID NO: 2); etc. etc. etc. . . .

[0176] In another aspect, C-terminal deletion mutants are provided bythe present invention. Preferably, the N-terminal amino acid residue ofsaid C-terminal deletion mutants is amino acid residue 1 (Met), 36(Thr), or 37 (Cys) of FIG. 1 (SEQ ID NO: 2). Such mutants include thosecomprising the amino acid sequence shown in FIG. 1 (SEQ ID NO: 2) exceptfor a deletion of at least the last C-terminal amino acid residue (Ser(208)) but not more than the last 55 C-terminal amino acid residues(i.e., a deletion of amino acid residues Glu (154)-Ser (208)) of FIG. 1(SEQ ID NO: 2). Alternatively, the deletion will include at least thelast C-terminal amino acid residue but not more than the last 65C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2). Alternatively,the deletion will include at least the last 10 C-terminal amino acidresidues but not more than the last 55 C-terminal amino acid residues ofFIG. 1 (SEQ ID NO: 2). Alternatively, the deletion will include at leastthe last 20 C-terminal amino acid residues but not more than the last 55C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2). Alternatively,the deletion will include at least the last 30 C-terminal amino acidresidues but not more than the last 55 C-terminal amino acid residues ofFIG. 1 (SEQ ID NO: 2). Alternatively, the deletion sill include at leastthe last 40 C-terminal amino acid residues but not more than the last 55C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2). Alternatively,the deletion will include at least the last 50 C-terminal amino acidresidues but not more than the last 55 C-terminal amino acid residues ofFIG. 1 (SEQ ID NO: 2).

[0177] In addition to the ranges of C-terminal deletion mutantsdescribed above, the present invention is also directed to allcombinations of the above described ranges, e.g., deletions of at leastthe last C-terminal amino acid residue but not more than the last 10C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2); deletions of atleast the last C-terminal amino acid residue but not more than the last20 C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2); deletions ofat least the last C-terminal amino acid residue but not more than thelast 30 C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2);deletions of at least the last C-terminal amino acid residue but notmore than the last 40 C-terminal amino acid residues of FIG. 1 (SEQ IDNO: 2); deletions of at least tile last 10 C-terminal amino acidresidues but not more than the last 20 C-terminal amino acid residues ofFIG. 1 (SEQ ID NO: 2); deletions of at least the last 10 C-terminalamino acid residues but not more than the last 30 C-terminal amino acidresidues of FIG. 1 (SEQ ID NO: 2); deletions of at least the last 10C-terminal amino acid residues but not more than the last 40 C-terminalamino acid residues of FIG. 1 (SEQ ID NO: 2); deletions of at least thelast 20 C-terminal amino acid residues but not more than the last 30C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2); etc. etc. etc.. . .

[0178] In yet another aspect, also included by the present invention aredeletion mutants having amino acids deleted from both the N-terminal andC-terminal residues. Such mutants include all combinations of theN-terminal deletion mutants and C-terminal deletion mutants describedabove. Such mutants include those comprising the amino acid sequenceshown in FIG. 1 (SEQ ID NO: 2) except for a deletion of at least thefirst 46 N-terminal amino acid residues but not more than the first 137N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2) and a deletionof at least the last C-terminal amino acid residue but not more than thelast 55 C-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2).Alternatively, a deletion can include at least the first 62, 68, 76, 92,103, or 122 N-terminal amino acids but not more than the first 137N-terminal amino acid residues of FIG. 1 (SEQ ID NO: 2) and a deletionof at least the last 10, 20, 30, 40, or 50 C-terminal amino acidresidues but not more than the last 55 C-terminal amino acid residues ofFIG. 1 (SEQ ID NO: 2). Further included are all combinations of theabove described ranges.

[0179] Substitution of Amino Acids

[0180] A further aspect of the present invention also includes thesubstitution of amino acids. Native mature KGF-2 contains 44 chargedresidues, 32 of which carry a positive charge. Depending on the locationof such residues in the protein's three dimensional structure,substitution of one or more of these clustered residues with amino acidscarrying a negative charge or a neutral charge may alter theelectrostatic interactions of adjacent residues and may be useful toachieve increased stability and reduced aggregation of the protein.Aggregation of proteins cannot only result in a loss of activity but beproblematic when preparing pharmaceutical formulations, because they canbe immunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967),Robbins et al., Diabetes 36: 838-845 (1987), Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10: 307-377 (1993)). Any modificationshould give consideration to minimizing charge repulsion in the tertiarystructure of the protein molecule. Thus, of special interest aresubstitutions of charged amino acid with another charge and with neutralor negatively charged amino acids. The latter results in proteins with areduced positive charge to improve the characteristics of KGF-2. Suchimprovements include increased stability and reduced aggregation of theanalog as compared to the native KGF-2 protein.

[0181] The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361: 266-268(1993), described certain TNF alpha mutations resulting in selectivebinding of TNF alpha to only one of the two known TNF receptors.

[0182] KGF-2 molecules may include one or more amino acid substitutions,deletions or additions, either from natural mutation or humanmanipulation. Examples of some preferred mutations are: Ala (49) Gln,Asn (51) Ala, Ser (54) Val, Ala (63) Pro, Gly (64) Glu, Val (67) Thr,Trp (79) Val, Arg (80) Lys, Lys (87) Arg, Tyr (88) Trp, Phe (89) Tyr,Lys (91) Arg, Ser (99) Lys, Lys (102) Gin, Lys 103(Glu), Glu (104) Met,Asn (105) Lys, Pro (107) Asn, Ser (109) Asn, Leu (111) Met, Thr (114)Arg, Glu(117) Ala, Val (120) Ile, Val (123) Ile, Ala (125) Gly, Ile(126) Val, Asn (127) Glu, Asn (127) Gln, Tyr (130) Phe, Met (134) Thr,Lys (136) Glu, Lys (137) Glu, Gly (142) Ala, Ser (143) Lys, Phe (146)Ser, Asn (148) Glu, Lys (151) Asn, Leu (152) Phe, Glu (154) Gly, Glu(154) Asp, Arg (155) Leu, Glu (157) Leu, Gly (160) His, Phe (167) Ala,Asn (168) Lys, Gln (170) Thr, Arg (174) Gly, Tyr (177) Phe, Gly (182)Gln, Ala (185) Val, Ala (185) Leu, Ala (185) Ile, Arg (187) Gln (190)Lys, Lys (195) Glu, Thr (197) Lys, Ser (198) Thr, Arg (194) Glu, Arg(194) Gln, Lys (191) Glu, Lys (191) Gln, Arg (188) Glu, Arg (188) Gln,Lys (183) Glu.

[0183] By the designation, for example, Ala (49) Gln is intended thatthe Ala at position 49 of FIG. 1 (SEQ ID NO: 2) is replaced by Gln.

[0184] Changes are preferably of minor nature, such as conservativeamino acid substitutions that do not significantly affect the folding oractivity of the protein. Examples of conservative amino acidsubstitutions known to those skilled in the art are set forth below:Aromatic: phenylalanine tryptophan tyrosine Hydrophobic: leucineisoleucine valine Polar: glutamine asparagine Basic: arginine lysinehistidine Acidic: aspartic acid glutamic acid Small: alanine serinethreonine methionine glycine

[0185] Of course, the number of amino acid substitutions a skilledartisan would make depends on many factors, including those describedabovre. Generally speaking, the number of substitutions for any givenKGF-2 polypeptide will not be more than 50, 40, 30, 20, 10, 5, or 3,depending on the objective. For example, a number of substitutions thatcan be made in the C-terminus of KGF-2 to improve stability aredescribed above and in Example 22.

[0186] Amino acids in KGF-2 that are essential for function can beidentified by methods well known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells,Science 244 :1081-1085 (1989). The latter procedure introduces singlealanine mutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity such as receptorbinding or in vitro and in vivo proliferative activity. (See, e.g.,Examples 10 and 11). Sites that are critical for ligand-receptor bindingcan also be determined by structural analyzis such as crystalization,nuclear magnetic resonance or photoaffinity labelling. (See for example:Smith et al., J. Mol. Biol., 224: 899-904 (1992); and de Vos et al.Science, 255: 306-312 (1992).)

[0187] Another aspect of the present invention substitutions of serinefor cysteine at amino acid positions 37 and 106 and 150. An unevennumber of cysteins means that at least one cysteine residue is availablefor intermolecular crosslinks or bonds that can cause the protein toadopt an undesirable tertiary structure. Novel KGF-2 proteins that haveone or more cysteine replaced by serine or e.g. alanine are generallypurified at a higher yield of soluble, correctly folded protein.Although not proven, it is believed that the cysteine residue atposition 106 is important for function. This cysteine residue is highlyconserved among all other FGF family members.

[0188] A further aspect of the present invention are fusions of KGF2with other proteins or fragments thereof such as fusions or hybrids withother FGF proteins, e.g. KGF (FGF-7), bFGF, aFGF, FGF-5, FGF-6, etc.Such a hybrid has been reported for KGF (FGF-7). In the published PCTapplication no. 90/08771 a chimeric protein has been produced consistingof the first 40 amino acid residues of KGF and the C-terminal portion ofAFGF. The chimera has been reported to target keratinocytes like KGF,but lacked suceptibility to heparin, a characteristic of aFGF but notKGF. Fusions with parts of the constant domain of immunoglobulins (IgG)show often an increased halflife time in vivo. This has been shown,e.g., for chimeric proteins consisting of the first two domains of thehuman CD4-polypeptide with various domains of the constant regions ofthe heavy or light chains of mammalian immunoglobulins (European Patentapplication, Publication No. 394 827, Traunecker et al., Nature, 331,84-86 (1988). Fusion proteins that have a disulfide-linked dimericstructure can also be more efficient in binding monomeric moleculesalone (Fountoulakis et al., J. of Biochemistry, 270: 3958-3964, (1995)).

[0189] Antigenic/hydrophilic Parts of KGF-2

[0190] As demonstrated in FIG. 4A-4E, there are 4 major highlyhydrophilic regions in the KGF-2 protein. Amino acid residuesGly41-Asn71, Lys91-Ser 109, Asn135-Tyr 164 and Asn 181-Ala 199 [SEQ IDNOS: 25-28]. There are two additional shorter predicted antigenic areas,Gln 74-Arg 78 of FIG. 1 (SEQ ID NO: 2) and Gln 170-Gln 175 of FIG. 1(SEQ ID NO: 2). Hydrophilic parts are known to be mainly at the outside(surface) of proteins and, therefore, available for antibodiesrecognizing these regions. Those regions are also likely to be involvedin the binding of KGF-2 to its receptor(s). Synthetic peptides derivedfrom these areas can interfere with the binding of KGF-2 to itsreceptor(s) and, therefore, block the function of the protein. Syntheticpeptides from hydrophilic parts of the protein may also be agonistic,i.e. mimic the function of KGF-2.

[0191] Thus, the present invention is further directed to isolatedpolypeptides comprising a hydrophilic region of KGF-2 wherein saidpolypeptide is not more than 150 amino acids in length, preferably notmore than 100, 75, or 50 amino acids in length, which comprise one ormore of the above described KGF-2 hydrophilic regions.

[0192] Chemical Modifications

[0193] The KGF wild type and analogs may be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties may improve the solubility, the biological halflife or absorption of the protein. The moieties may also reduce oreliminate any desirable side effects of the proteins and the like. anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 18th ed., Mack Publishing Co., Easton, Pa. (1990).Polyethylene glycol (PEG) is one such chemical moiety which has beenused for the preparation of therapeutic proteins. The attachment of PEGto proteins has been shown to protect against proteolysis, Sada et al.,J. Fermentation Bioengineering 71: 137-139 (1991). Various methods areavailable for the attachment of certain PEG moieties. For review, see:Abuchowski et al., in Enzymes as Drugs. (Holcerberg and Roberts, eds.)pp. 367-383 (1981). Many published patents describe derivatives of PEGand processes how to prepare them, e.g., Ono et al. U.S. Pat. No.5,342,940; Nitecki et al. U.S. Pat. No. 5,089,261; Delgado et al. U.S.Pat. No. 5,349,052. Generally, PEG molecules are connected to theprotein via a reactive group found on the protein. Amino groups, e.g. onlysines or the amino terminus of the protein are convenient for thisattachment among others.

[0194] The entire disclosure of each document cited in this section on“Polypeptides and Peptides” is hereby incorporated herein by reference.

[0195] Vectors and Host Cells

[0196] The present invention also relates to vectors which include theisolated DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof KGF-2 polypeptides or fragments thereof by recombinant techniques.

[0197] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention. The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0198] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the KGF-2 genes. The culture conditions,such as temperature, pH and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

[0199] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0200] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0201] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequences) (promoter) to directcDNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0202] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0203] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0204] As representative examples of appropriate hosts, there may bementioned:

[0205] bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimirium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0206] In addition to the use of expression vectors in the practice ofthe present invention, the present invention further includes novelexpression vectors comprising operator and promoter elements operativelylinked to nucleotide sequences encoding a protein of interest. Oneexample of such a vector is pHE4-5 which is described in detail below.

[0207] As summarized in FIGS. 50 and 51, components of the pHE4-5 vector(SEQ ID NO: 147) include: 1) a neomycinphosphotransferase gene as aselection marker, 2) an E. coli origin of replication, 3) a T5 phagepromoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarnosequence, 6) the lactose operon repressor gene (lacIq). The origin ofreplication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). Thepromoter sequence and operator sequences were made synthetically.Synthetic production of nucleic acid sequences is well known in the art.CLONTECH 95/96 Catalog, pages 215-216, CLONTECH, 1020 East MeadowCircle, Palo Alto, Calif. 94303. A nucleotide sequence encoding KGF-2(SEQ ID NO: 1), is operatively linked to the promoter and operator byinserting the nucleotide sequence between the NdeI and Asp718 sites ofthe pHE4-5 vector.

[0208] As noted above, the pHE4-5 vector contains a lacIq gene. LacIq isan allele of the lacI gene which confers tight regulation of the lacoperator. Amann, E. et al., Gene 69:301-315 (1988); Stark, M., Gene51:255-267 (1987). The lacIq gene encodes a repressor protein whichbinds to lac operator sequences and blocks transcription of down-stream(i.e., 3′) sequences. However, the lacIq gene product dissociates fromthe lac operator in the presence of either lactose or certain lactoseanalogs, e.g., isopropyl B-D-thiogalactopyranoside (IPTG). KGF-2 thus isnot produced in appreciable quantities in uninduced host cellscontaining the pHE4-5 vector. Induction of these host cells by theaddition of an agent such as IPTG, however, results in the expression ofthe KGF-2 coding sequence.

[0209] The promoter/operator sequences of the pHE4-5 vector (SEQ ID NO:148) comprise a T5 phage promoter and two lac operator sequences. Oneoperator is located 5′ to the transcriptional start site and the otheris located 3′ to the same site. These operators, when present incombination with the lacIq gene product, confer tight repression ofdown-stream sequences in the absence of a lac operon inducer, e.g.,IPTG. Expression of operatively linked sequences located down-streamfrom the lac operators may be induced by the addition of a lac operoninducer, such as IPTG. Binding of a lac inducer to the lacIq proteinsresults in their release from the lac operator sequences and theinitiation of transcription of operatively linked sequences. Lac operonregulation of gene expression is reviewed in Devlin, T., TEXTBOOK OFBIOCHEMISTRY WITH CLINICAL CORRELATIONS, 4th Edition (1997), pages802-807.

[0210] The pHE4 series of vectors contain all of the components of thepHE4-5 vector except for the KGF-2 coding sequence. Features of the pHE⁴vectors include optimized synthetic T5 phage promoter, lac operator, andShine-Delagamo sequences. Further, these sequences are also optimallyspaced so that expression of an inserted gene may be tightly regulatedand high level of expression occurs upon induction.

[0211] Among known bacterial promoters suitable for use in theproduction of proteins of the present invention include the E. coli lacIand lacZ promoters, the T3 and T7 promoters, the gpt promoter, thelambda PR and PL promoters and the trp promoter. Suitable eukaryoticpromoters include the CMV immediate early promoter, the HSV thymidinekinase promoter, the early and late SV40 promoters, the promoters ofretroviral LTRs, such as those of the Rous Sarcoma Virus (RSV), andmetallothionein promoters, such as the mouse metillothionein-I promoter.

[0212] The pHE4-5 vector also contains a Shine-Delgarno sequence 5′ tothe AUG initiation codon. Shine-Delgarno sequences are short sequencesgenerally located about 10 nucleotides up-stream (Le., 5′) from the AUGinitiation codon. These sequences essentially direct prokaryoticribosomes to the AUG initiation codon.

[0213] Thus, the present invention is also directed to expression vectoruseful for the production of the proteins of the present invention. Thisaspect of the invention is exemplified by the pHE4-5 vector (SEQ ID NO:147). The pHE4-5 vector containing a cDNA insert encoding KGF-2 Δ33 wasdeposited at the ATCC on Jan. 9, 1998 as ATCC No. 209575.

[0214] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9((Qiagen), pBS, pDlO, phagescript, psiX174, pbluescript SK, pbsks,pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any otherplasmid or vector may be used as long as they are replicable and viablein the host.

[0215] Promoter regions can be selected from any desired gene, using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0216] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfetion, or electroporation (Davis, L. et al., BasicMethods in Molecular Biology (1986)).

[0217] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0218] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989), the disclosure of which is hereby incorporated byreference.

[0219] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovimis early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0220] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

[0221] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals, but alsoadditional heterologous functional regions. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizereceptors. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc pat after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,shIL5- has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition, Vol. 8 52-58 (1995)and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270,No. 16, pp 9459-9471 (1995).

[0222] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0223] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0224] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA) These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0225] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0226] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0227] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well known to those skilled in the art.

[0228] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzmar, Cell 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0229] The KGF-2 polypeptide can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0230] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0231] Diagnostic and Therapeutic Applications of KGF-2

[0232] As used in the section below, “KGF-2” is intended to refer to thefull-length and mature forms of KGF-2 described herein and to the KGF-2analogs, derivatives and mutants described herein. This invention isalso related to the use of the KGF-2 gene as part of a diagnostic assayfor detecting diseases or susceptibility to diseases related to thepresence of mutations in the KGF-2 nucleic acid sequences.

[0233] Individuals carrying mutations in the KGF-2 gene may be detectedat the DNA level by a variety of techniques. Nucleic acids for diagnosismay be obtained from a patient's cells, such as from blood, urine,saliva, tissue biopsy and autopsy material. The genomic DNA may be useddirectly for detection or may be amplified enzymatically by using PCR(Saiki et al., Nature 324:163-166 (1986)) prior to analysis. RNA or cDNAmay also be used for the same purpose. As an example, PCR primerscomplementary to the nucleic acid encoding KGF-2 can be used to identifyand analyze KGF-2 mutations. For example, deletions and insertions canbe detected by a change in size of the amplified product in comparisonto the normal genotype. Point mutations can be identified by hybridizingamplified DNA to radiolabeled KGF-2 RNA or alternatively, radiolabeledKGF-2 antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

[0234] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel ectrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0235] Sequence changes at specific locations may also be revealed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0236] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA

[0237] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0238] The present invention also relates to a diagnostic assay fordetecting altered levels of KGF-2 protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease, for example, a tumor. Assays used to detect levels of KGF-2protein in a sample derived from a host are well-known to those of skillin the art and include radioimmunoassays, competitive-binding assays,Western Blot analysis, ELISA assays and “sandwich” assay. An ELISA assay(Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6,(1991)) initially comprises preparing an antibody specific to the KGF-2antigen, preferably a monoclonal antibody. In addition a reporterantibody is prepared against the monoclonal antibody. To the reporterantibody is attached a detectable reagent such as radioactivity,fluorescence or, in this example, a horseradish peroxidase enzyme. Asample is removed from a host and incubated on a solid support, e.g. apolystyrene dish, that binds the proteins in the sample. Any freeprotein binding sites on the dish are then covered by incubating with anon-specific protein like bovine serum albumen. Next, the monoclonalantibodies attach to any KGF-2 proteins attached to the polytyrene dish.All unbound monoclonal antibody is washed out with buffer. The reporterantibody linked to horseradish peroxidase is now placed in the dishresulting in binding of the reporter antibody to any monoclonal antibodybound to KGF-2. Unattached reporter antibody is then washed out.Peroxidase substrates are then added to the dish and the amount of colordeveloped in a given time period is a measurement of the amount of KGF-2protein present in a given volume of patient sample when comparedagainst a standard curve.

[0239] A competition assay may be employed wherein antibodies specificto KGF-2 are attached to a solid support and labeled KGF-2 and a samplederived from the host are passed over the solid support and the amountof label detected, for example by liquid scintillation chromatography,can be correlated to a quantity Of KGF-2 in the sample.

[0240] A “sandwich” assay is similar to an ELISA assay. In a “sandwich”assay KGF-2 is passed over a solid support and binds to antibodyattached to a solid support. A second antibody is then bound to theKGF-2. A third antibody which is labeled and specific to the secondantibody is then passed over the solid support and binds to the secondantibody and an amount can then be quantified.

[0241] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single, chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0242] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0243] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler & Milstein,Nature, 256:495-497 (1975)), the trioma technique, the human B-cellhybridoma technique (Kozbor, et al., Immunology Today 4:72 (1983)), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96 (1985)).

[0244] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0245] The polypeptides of the present invention may be employed tostimulate new blood vessel growth or angiogenesis. Particularly, thepolypeptides of the present invention may stimulate keratinocyte cellgrowth and proliferation. Accordingly the present invention provides aprocess for utilizing such polypeptide, or polynucleotide encoding suchpolypeptide for therapeutic purposes, for example, to stimulateepithelial cell proliferation and basal keratinocytes for the purpose ofwound healing, and to stimulate hair follicle production and healing ofdermal wounds.

[0246] As noted above, the polypeptides of the present invention may beemployed to heal dermal wounds by stimulating epithelial cellproliferation. These wounds may be of superficial nature or may be deepand involve damage of the dermis and the epidermis of skin. Thus, thepresent invention provides a method for the promotion of wound healingthat involves the administration of an effective amount of KGF-2 to anindividual.

[0247] The individual to which KGF-2 is administered may heal wounds ata normal rate or may be healing impaired. When administered to anindividual who is not healing impaired, KGF-2 is administered toaccelerate the normal healing process. When administered to anindividual who is healing impaired, KGF-2 is administered to facilitatethe healing of wounds which would otherwise heal slowly or not at all.As noted below, a number of afflictions and conditions can result inhealing impairment. These afflictions and conditions include diabetes(e.g., Type II diabetes mellitus), treatment with both steroids andother pharmacological agents, and ischemic blockage or injury. Steroidswhich have been shown to impair wound healing include cortisone,hydrocortisone, dexamethasone, and methylprednisolone.

[0248] Non-steroid compounds, e.g., octreotide acetate, have also beenshown to impair wound healing. Waddell, B. et al., Am. Surg. 63:446-449(1997). The present invention is believed to promote wound healing inindividuals undergoing treatment with such non-steroid agents.

[0249] A number of growth factors have been shown to promote woundhealing in healing impaired individuals. See, e.g., Steed, D. et al., J.Am. Coll. Surg. 183:61-64 (1996); Richard, J. et al., Diabetes Care 18:64-69 (199,5); Steed, D., J. Vasc. Surg. 21:71-78 (1995); Kelley, S. etal., Proc. Soc. Exp. Biol 194:320-326 (1990). These growth factorsinclude growth hormone-releasing factor, platelet-derived growth factor,and basic fibroblast growth factor. Thus, the present invention alsoencompasses the administration of KGF-2 in conjunction with one or moreadditional growth factors or other agent which promotes wound healing.

[0250] The present invention also provides a method for promoting thehealing of anastomotic and other wounds caused by surgical procedures inindividuals which both heal wounds at a normal rate and are healingimpaired. This method involves the administration of an effective amountof KGF-2 to an individual before, after, and/or during anastomotic orother surgery. Anastomosis is the connecting of two tubular structures,as which happens, for example, when a mid-section of intestine isremoved and the remaining portions are linked together to reconstitutethe intestinal tract. Unlike with cutaneous healing, the healing processof anastomotic wounds is generally obscured from view. Further, woundhealing, at least in the gastrointestinal tract, occurs rapidly in theabsence of complications; however, complications often requirecorrection by additional surgery. Thornton, F. and Barbul, A., Surg.Clin. North Am. 77:549-573 (1997). As shown in Examples 21 and 28,treatment with KGF-2 causes a significant decrease in peritoneal leakageand anastomotic constriction following colonic anastomosis. KGF-2 isbelieved to cause these results by accelerating the healing process thusdecreasing the probability of complications arising following suchprocedures.

[0251] Thus, the present invention also provides a method foraccelerating healing after anastomoses or other surgical procedures inan individual, which heals wounds at a normal rate or is healingimpaired, compromising the administration of an effective amount ofKGF-2.

[0252] The polypeptides of the present invention may also be employed tostimulate differentiation of cells, for example muscle cells, cellswhich make up nervous tissue, prostate cells, and lung cells.

[0253] KGF-2 may be clinically useful in stimulating wound healing ofwounds including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, and burnsresulting from heat exposure or chemicals, in normal individuals andthose subject to conditions which induce abnormal wound healing such asuremia, malnutrition, vitamin deficiencies, obesity, infection,immunosuppression and complications associated with systemic treatmentwith steroids, radiation therapy, and antineoplastic drugs andantimetabolites. KGF-2 is also useful for promoting the healing ofwounds associated with ischemia and ischemic injury, e.g., chronicvenous leg ulcers caused by an impairment of venous circulatory systemreturn and/or insufficiency.

[0254] KGF-2 can also be used to promote dermal reestablishmentsubsequent to dermal loss. In addition, KGF-2 can be used to increasethe tensile strength of epidermis and epidermal thickness.

[0255] KGF-2 can be used to increase the adherence of skin grafts to awound bed and to stimulate re-epithelialization from the wound bed. Thefollowing are types of grafts that KGF-2 could be used to increaseadherence to a wound bed: autografts, artificial skin, allografts,autodermic graft, autoepidermic grafts, avacular grafts, Blair-Browngrafts, bone graft, brephoplastic grafts, cutis graft, delayed graft,dermic graft, epidermic graft, fascia graft, full thickness graft,heterologous graft, xenograft, homologous graft, hyperplastic graft,lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft,omenpal graft, patch graft, pedicle graft, penetrating graft, split skingraft, thick split graft. KGF-2 can be used to promote skin strength andto improve the appearance of aged skin.

[0256] It is believed that KGF-2 will also produce changes in hepatocyteproliferation, and epithelial cell proliferation in the lung, breast,pancreas, stomach, small intestine, and large intestine. KGF-2 canpromote proliferation of epithelial cells such as sebocytes, hairfollicles, hepatocytes, type II pneumocytes, mucin-producing gobletcells, and other epithelial cells and their progenitors contained withinthe skin, lung, liver, kidney and gastrointestinal tract. As shown inExample 31, KGF-2 stimulates the proliferation of hepatocytes. Thus,KGF-2 can also be used prophylactically or therapeutically to prevent orattenuate acute or chronic viral hepatitis as well as fulminant orsubfulminant liver failure caused by diseases such as acute viralhepatitis, cirrhosis, drug- and toxin-induced hepatitis (e.g,acetaminophen, carbon tetrachloride, methotrexate, organic arsenicals,and other hepatotoxins known in the art), autoimmune chronic activehepatitis, liver transplantation, and partial hepatectomy (Cotran et al.Pathologic basis of disease. (51 ed). Philadelphia, W. B. SaundersCompany, 1994). KGF-2 can also be used to stimulate or promote liverregeneration and in patients with alcoholic liver disease.

[0257] Approximately 80% of acute pancreatitis cases are associated withbiliary tract disease and alcoholism (Rattner D. W., Scand JGastroenterol 31:6-9 (1996); Cotran et al. Pathologic basis of disease.(5^(th) ed). Philadelphia, W.B. Saunders Company, 1994). Acutepancreatitis is an important clinical problem with significant morbidityand mortality (Banerjee et al., British Journal of Surgery 81:1096-1103(1994)). The pathogenesis of this disease is still somewhat unresolvedbut it is widely recognized that pancreatic enzymes are released withinthe pancreas leading to proteolysis, interstitial inflammation, fatnecrosis, and hemorrhage. Acute pancreatitis can lead to disseminatedintravascular coagulation, adult respiratory distress syndrom, shock,and acute renal tubular necrosis (Cotran et al. Pathologic basis ofdisease. (5^(th) ed). Philadelphia, W.B. Saunders Company, 1994).Despite palliative measures, about 5% of these patients die of shockduring the first week of the clinical course. In surviving patients,sequelae may include pancreatic abscess, pseudocyst, and duodenalobstruction (Cotran et al. Pathologic basis of disease. (5^(th) ed).Philadelphia, W.B. Saunders Company, 1994). Chronic pancreatitis isoften a progressive destruction of the pancreas caused by repeatedflare-ups of acute pancreatitis. Chronic pancreatitis appears to incur amodestly increased risk of pancreatic carcinoma (Cotran et al.Pathologic basis of disease. (5^(th) ed). Philadelphia, W.B. SaundersCompany, 1994).

[0258] As indicated above and in Example 31, KGF-2 also promotesproliferation of pancreatic cells. Thus, in a further aspect, KGF-2 canbe used prophylactically or therapeutically to prevent or attenuateacute or chronic pancreatitis.

[0259] KGF-2 can also be used to reduce the side effects of gut toxicitythat result from the treatment of viral infections, radiation therapy,chemotherapy or other treatments. KGF-2 may have a cytoprotective effecton the small intestine mucosa. KGF-2 may also be used prophylacticallyor therapeutically to prevent or attenuate mucositis and to stimulatehealing of mucositis (e.g., oral, esophageal, intestinal, colonic,rectal, and anal ulcers) that result from chemotherapy, other agents andviral infections. Thus the present invention also provides a method forpreventing or treating diseases or pathological events of the mucosa,including ulcerative colitis, Crohn's disease, and other diseases wherethe mucosa is damaged, comprising the administration of an effectiveamount of KGF-2. The present invention similarly provides a method forpreventing or treating oral (including odynophagia associated withmucosal injury in the pharynx and hypopharynx), esophageal, gastric,intestinal, colonic and rectal mucositis irrespective of the agent ormodality causing this damage.

[0260] KGF-2 can promote proliferation of endothelial cells,keratinocytes, and basal keratinocytes. Thus, the present invention alsoprovides a method for stimulating the proliferation of such cell typeswhich involves contacting cells with an effective amount of KGF-2. KGF-2may be administered to an individual in an effective amount to stimulatecell proliferation in vivo or KGF-2 may be contacted with such cells invitro.

[0261] The present invention further provides a method for promotingurothelial healing comprising administering an effective amount of KGF-2to an individual. Thus, the present invention provides a method foraccelerating the healing or treatment of a variety of pathologiesinvolving urothelial cells (i.e., cells which line the urinary tract).Tissue layers comprising such cells may be damaged by numerousmechanisms including catheterization, surgery, or bacterial infection(e.g., infection by an agent which causes a sexually transmitteddisease, such as gonorrhea).

[0262] The present invention also encompasses methods for the promotionof tissue healing in the female genital tract comprising theadministration of an effective amount of KGF-2. Tissue damage in thefemale genital tract may be caused by a wide variety of conditionsincluding Candida infections trichomoniasis, Gardnerella, gonorrhea,chlamydia, mycoplasma infections and other sexually transmitteddiseases.

[0263] As shown in Examples 10, 18, and 19 KGF-2 stimulates theproliferation of epidermal keratinocytes and increases epidermalthickening. Thlis, KGF-2 can be used in full regeneration of skin; infull and partial thickness skin defects, including burns (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands); andthe treatment of other skin defects such as psoriasis.

[0264] KGF-2 can be used to treat epidermolysis bullosa, a defect inadherence of the epidermis to the underlying dermis which results infrequent, open and painful blisters by accelerating reepithelializationof these lesions. KGF-2 can also be used to treat gastric and duodenalulcers and help heal the mucosal lining and regeneration of glandularmucosa and duodenal mucosal lining more rapidly. Inflammatory boweldiseases, such as Crohn's disease and ulcerative, colitis, are diseaseswhich result in destruction of the mucosal surface of the small or largeintestine, respectively. Thus, KGF-2 could be used to promote theresurfacing of the mucosal surface to aid more rapid healing and toprevent or attenuate progression of inflammatory bowel disease. KGF-2treatment is expected to have a significant effect on the production ofmucus throughout the gastrointestinal tract and could be used to protectthe intestinal mucosa from injurious substances that are ingested orfollowing surgery. As noted above, KGF-2 can also be used to promotehealing of intestinal or colonic anastomosis. KGF-2 can further be usedto treat diseases associate with the under expression of KGF-2.

[0265] As shown in Example 32 below, KGF-2 stimulates proliferation oflung epithelial cells. Thus, KGF-2 can be administered prophylacticallyto reduce or prevent damage to the lungs caused by various pathologicalstates. KGF-2 can also be administered during or after a damaging eventoccurs to prontote healing. For example, KGF-2 can stimulateproliferation and differentiation and promote the repair of alveoli andbronchiolar epithelium to prevent, attenuate, or treat acute or chroniclung damage. Emphysema, which results in the progressive loss ofalveoli, and inhalation injuries, i.e., resulting from smoke inhalationand burns, that cause necrosis of the bronchiolar epithelium and alveolicould be effectively treated using KGF-2 as could damage attributable tochemotherapy, radiation treatment, lung cancer, asthma, black lung andother lung damaging conditions. Also, KGF-2 could be used to stimulatethe proliferation of and differentiation of type II pneumocytes, whichmay help treat or prevent disease such as hyaline membrane diseases,such as infant respiratory distress syndrome and bronchopulmonarydysplasia, in premature infants.

[0266] The three causes of acute renal failure are prerenal (e.g., heartfailure), intrinsic (e.g., nephrotoxicity induced by chemotherapeuticagents) and postrenal (e.g., urinary tract obstruction) which lead torenal tubular cell death, obstruction of the tubular lumens, and backflow of filtrate into the glomeruli (reviewed by Thadhani et al. N.Engl. J. Med. 334:1448-1460 (1996)). Growth factors such as insulin-likegrowth factor I, osteogenic protein-1, hepatocyte growth factor, andepidermal growth factor have shown potential for ameliorating renaldisease in animal models. Taub et al. Cytokine 5:175-179 (1993);Vukicevic et al. J. Am. Soc. Nephrol. 7:1867 (1996). As shown in Example31 below, KGF-2 stimulates proliferation of renal epithelial cells and,thus, is useful for alleviating or treating renal diseases andpathologies such as acute and chronic renal failure and end stage renaldisease.

[0267] KGF-2 could stimulate the proliferation and differentiation ofbreast tissue and therefor could be used to promote healing of breasttissue injury due to surgery, trauma, or cancer.

[0268] In addition, KGF-2 could be used treat or prevent the onset ofdiabetes mellitus. In patients with newly diagnosed Types I and IIdiabetes, where some islet cell function remains, KGF-2 could be used tomaintain the islet function so as to alleviate, delay or preventpermanent manifestation of the disease. Also, KGF-2 could be used as anauxiliary in islet cell transplantation to improve or promote islet cellfunction.

[0269] Further, the anti-inflammatory property of KGF-2, could bebeneficial for treating acute and chronic conditions in whichinflammation is a key pathogenesis of the diseases including, but notlimiting to, psoriasis, eczema, dermiatitis and/or arthritis. Thus, thepresent invention provides a method for preventing or attenuatinginflammation, and diseases involving inflammation, in an individualcomprising the administration of an effective amount of KGF-2.

[0270] KGF-2 can be used to promote healing and alleviate damage ofbrain tissue due to injury from trauma, surgery or chemicals.

[0271] In addition, since KGF-2 increases the thickness of theepidermis, the protein could be used for improving aged skin, reducingwrinkles in skin, reducing scarring after surgery. Scarring of woundtissues often involves hyperproliferation of dermal fibroblasts. Asnoted in Example 10, fibroblast proliferation is not stimulated byKGF-2. Therefore, KGF-2 appeares to be a mitogen specific for epidermalkeratinocytes and induces wound healing with minimal scarring. Thus, thepresent invention provides a method for promoting the healing of woundswith minimal scarring involving the administration of an effectiveamount of KGF-2 to an individual. KGF-2 may be administered prior to,during, and/or after the process which produces the wound (e.g. cosmeticsurgery, accidental or deliberate tissue trauma caused by a sharpobject).

[0272] As noted above, KGF-2 also stimulates the proliferation ofkeratinocytes and hair follicles and therefore can be used to promotehair growth from balding scalp, and in hair transplant patients. Thus,the present invention further provides a method for promoting hairgrowth comprising the administration of an amount KGF-2 sufficient tostimulate the production of hair follicles.

[0273] The present invention also provides a method for protecting anindividual from the effects of ionizing radiation, chemotherapy, ortreatment with anti-viral agents comprising the administration of aneffective amount of KGF-2. The present invention further provides amethod for treating tissue damage which results from exposure toionizing radiation, chemotherapeutic agents, or anti-viral agentscomprising the administration of an effective amount of KGF-2. Anindividual may be exposed to ionizing radiation for a number of reasons,including for therapeutic purposes (e.g., for the treatment ofhyperproliferative disorders), as the result of an accidental release ofa radioactive isotope into the environment, or during non-invasivemedical diagnostic procedures (e.g., X-rays). Further, a substantialnumber of individuals are exposed to radioactive radon in their workplaces and homes. Long-term continuous environmental exposure has beenused to calculate estimates of lost life expectancy. Johnson, W. andKearfott, K., Health Phys. 73:312-319 (1997). As shown in Example 23,the proteins of the present invention enhance the survival of animalsexposed to radiation. Thus, KGF-2 can be used to increase survival rateof individuals suffering radiation-induced injuries, to protectindividuals from sub-lethal doses of radiation, and to increase thetherapeutic ratio of irradiation in the treatment of afflictions such ashyperproliferative disorders.

[0274] KGF-2 may also be used to protect individuals against dosages ofradiation, chemotherapeutic drugs or antiviral agents which normallywould not be tolerated. When used in this manner, or as otherwisedescribed herein, KGF-2 may be administered prior to, after, and/orduring radiation therapy/exposure, chemotherapy or treatment withanti-viral agents. High dosages of radiation and chemotherapeutic agentsmay be especially useful when treating an individual having an advancedstage of an afflication such as a hyperprolifereative disorder.

[0275] In another aspect, the present invention provides a method forpreventing or treating conditions such as radiation-induced oral andgastro-intestinal injury, mucositis, intestinal fibrosis, proctitis,radiation-induced pulmonary fibrosis, radiation-induced pneumonitis,radiation-induced pleural retraction, radiation-induced hemopoieticsyndrome, radiation-induced myelotoxicity, comprising administering aneffective amount of KGF-2 to an individual.

[0276] KGF-2 may be used alone or in conjunction with one or moreadditional agents which confer protection against radiation or otheragents. A number of cytokines (e.g., IL-1, TNF, IL-6, IL-12) have beenshown to confer such protective. See, e.g., Neta, R. et at., J. Exp.Med. 173:1177(1991). Additionally, IL-11 has been shown to protect smallintestinal mucosal cells after combined irradiation and chemotherapy,Du, X. X. et al., Blood 83:33 (1994), and radiation-induced thoracicinjury. Redlich, C. A. et al, J. Immun. 157:1705-1710 (1996). Severalgrowth factors have also been shown to confer protection to radiationexposure, e.g., fibroblast growth factor and transforming growth factorbeta-3. Ding, I. et al., Acta Oncol. 36:337-340 (1997); Potten, C. etal., Br. J. Cancer 75:1454-1459 (1997).

[0277] Hemorrhagic cystitis is a syndrome associated with certaindisease states as well as exposure to drugs, viruses, and toxins. Itmanifests as difuse bleeding of the endothelial lining of the bladder.Known treatments include intravesical, systemic, and nonpharmacologictherapies (West, N. J., Pharmacotherapy 17:696-706 (1997). Somecytotoxic agents used clinically have side effects resulting in theinhibition of the proliferation of the normal epithelial in the bladder,leading to potentially life-threatening ulceration and breakdown in theepithelial lining. For example, cyclophosphamide is a cytotoxic agentwhich is biotransformed principally in the liver to active alkylatingmetabolites by a mixed function microsomal oxidase system. Thesemetabolites interfere with the growth of susceptible rapidlyproliferating malignant cells. The mechanism of action is believed toinvolve cross-linking of tumor cell DNA (Physicians' Desk reference,1997).

[0278] Cyclophosphamide is one example of a cytotoxic agent which causeshemorrhagic cystitis in some patients, a complication which can besevere and in some cases fatal. Fibrosis of the urinary bladder may alsodevelop with or without cystitis. This injury is thought to be caused bycyclophosphamide metabolites excreted in the urine. Hematuria caused bycyclophosphamide usually is present for several days, but may persist.In severe cases medical or surgical treatment is required. Instances ofsevere hemorrhagic cystitis result in discontinued cyclophosphamidetherapy. In addition, urinary bladder malignancies generally occurwithin two years of cyclophosphamide treatment nd occurs in patients whopreviously had hemorrhagic cystitis (CYTOXAN (cyclophosphamide) packageinsert). Cyclophosphamide has toxic effects on the prostate and malereproductive systems. Cyclophosphamide treatment can result in thedevelopment of sterility, and result in some degree of testicularatrophy.

[0279] As shown in FIGS. 52 and 53, systemic administration of KGF-2 toan individual stimulates proliferation of bladder and prostaticepithelial cells. Thus, in one aspect, the present invention provides amethod of stimulating proliferation of bladder epithelium and prostaticepithelial cells by administering to an individual an effective amountof a KGF-2 polypeptide. More importantly, as FIGS. 54 and 55demonstrate, KGF-2 can be used to reduce damage caused by cytotoxicagents having side effects resulting in the inhibition of bladder andprostate epithelial cell proliferation. To reduce such damage, KGF-2 canbe administered either before, after, or during treament with orexposure to the cytotoxic agent. Accordingly, in a further aspect, thereis provided a method of reducing damage caused by an inhibition of thenormal proliferation of epithelial cells of the bladder or prostate byadministering to an individual an effective amount of KGF-2. Asindicated, inhibitors of normal proliferation of bladder or prostateepithelium include radiation therapy (causing acute or chronic radiationdamage) and cytotoxic agents such as chemotherapeutic or antineoplasticdrugs including, but not limited to, cyclophosphamide, busulfan, andifosfamide. In a further aspect, KGF-2 is administered to reduce orprevent fibrosis and ulceration of the urinary bladder. Preferably,KGF-2 is administered to reduce or prevent hemorrhagic cystitis.Suitable doses, formulations, and administration routes are describedbelow.

[0280] As used herein, by “individual” is intended an animal, preferablya mammal (such as apes, cows, horses, pigs, boars, sheep, rodents,goats, dogs, cats, chickens, monkeys, rabbits, ferrets, whales, anddolphins), and more preferably a human.

[0281] The signal sequence of KGF-2 encoding amino acids 1 through 35 or36 may be employed to identify secreted proteins in general byhybridization and/or computational search algorithms.

[0282] The nucleotide sequence of KGF-2 could be employed to isolate 5′sequences by hybridization. Plasmids comprising the KGF-2 gene under thecontrol of its native promoter/enhancer sequences could then be used inin vitro studies aimed at the identification of endogenous cellular andviral transactivators of KGF-2 gene expression.

[0283] The KGF-2 protein may also be employed as a positive control inexperiments designed to identify peptido-mimetics acting upon the KGF-2receptor.

[0284] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA, manufacture of DNAvectors and for the purpose of providing diagnostics and therapeuticsfor the treatment of human disease.

[0285] Fragments of the full length KGF-2 gene may be used as ahybridization probe for a cDNA library to isolate the full length KGF-2genes and to isolate other genes which have a high sequence similarityto these genes or similar biological activity. Probes of this typegenerally have at least 20 bases. Preferably, however, the probes haveat least 30 bases and generally do not exceed 50 bases, although theymay have a greater number of bases. The probe may also be used toidentify a cDNA clone corresponding to a full length transcript and agenomic clone or clones that contain the complete KGF-2 gene includingregulatory and promotor regions, exons, and introns. An example of ascreen comprises isolating the coding region of the KGF-2 gene by usingthe known DNA sequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to that of the gene ofthe present invention are used to screen a library of human cDNA,genomic DNA or cDNA to determine which members of the library the probehybridizes to.

[0286] This invention provides a method for identification of thereceptors for the KGF-2 polypeptide. The gene encoding the receptor canbe identified by numerous methods known to those of skill in the art,for example, ligand panning and FACS sorting (Coligan et al., CurrentProtocols in Immun., 1(2), Chapter 5 (1991)). Preferably, expressioncloning is employed wherein polyadenylated RNA is prepared from a cellresponsive to the polypeptides, and a cDNA library created from this RNAis divided into pools and used to transfect COS cells or other cellsthat are not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the labeled polypeptides. Thepolypeptides can be labeled by a variety of means including iodinationor inclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toautoradiogaphic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andrescreening process, eventually yielding a single clones that encodesthe putative receptor.

[0287] As an alternative approach for receptor identification, thelabeled polypeptides can be photoaffinity linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE analysis and exposed to x-ray film. Thelabeled complex containing the receptors of the polypeptides can beexcised, resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

[0288] This invention provides a method of screening compounds toidentify those which agonize the action of KGF-2 or block the functionof KGF-2. An example of such an assay comprises combining a mammalianKeratinocyte cell, the compound to be screened and ³[H] thymidine undercell culture conditions where the keratinocyte cell would normallyproliferate. A control assay may be performed in the absence of thecompound to be screened and compared to the amount of keratinocyteproliferation in the presence of the compound to determine if thecompound stimulates proliferation of Keratinocites.

[0289] To screen for antagonists, the same assay may be prepared in thepresence of KGF-2 and the ability of the compound to preventKeratinocyte proliferation is measured and a determination of antagonistability is made. The amount of Keratinocyte cell proliferation ismeasured by liquid scintillation chromatography which measures theincorporation of ³[H] thymidine.

[0290] In another method, a mammalian cell or membrane preparationexpressing the KGF-2 receptor would be incubated with labeled KGF-2 inthe presence of the compound. The ability of the compound to enhance orblock this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction ofKGF-2 and receptor would be measured and compared in the presence orabsence of the compound. Such second messenger systems include but arenot limited to, cAMP guanylate cyclase, ion channels or phosphoinositidehydrolysis.

[0291] Examples of potential KGF-2 antagonists include an antibody, orin some cases, an oligonucleotide, which binds to the polypeptide.Alternatively, a potential KGF-2 antagonist may be a mutant form ofKGF-2 which binds to KGF-2 receptors, however, no second messengerresponse is elicited and therefore the action of KGF-2 is effectivelyblocked.

[0292] Another potential KGF-2 antagonist is an antisense constructprepared using antisense technology. Antisense technology can be used tocontrol gene expression through triple-helix formation or antisense DNAor RNA, both of which methods are based on binding of a polynucleotideto DNA or RNA. For example, the 5′ coding portion of the polynucleotidesequence, which encodes for the mature polypeptides of the presentinvention, is used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcription(triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney etal., Science 241:456 (1988); and Dervan et al., Science 251:1360(1991)), thereby preventing transcription and the production of KGF-2.The antisense RNA oligonucleotide hybridizes to the cDNA in vivo andblocks translation of the cDNA molecule into KGF-2 polypeptide(Antisense-Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of KGF-2.

[0293] Potential KGF-2 antagonists include small molecules which bind toand occupy the binding site of the KGF-2 receptor thereby making thereceptor inaccessible to KGF-2 such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall peptides or peptide-like molecules.

[0294] The KGF-2 antagonists may be employed to prevent the induction ofnew blood vessel growth or angiogenesis in tumors. Angiogenesisstimulated by KGF-2 also contributes to several pathologies which mayalso be treated by the antagonists of the present invention, includingdiabetic retinopathy, and inhibition of the growth of pathologicaltissues, such as in rheumatoid arthritis.

[0295] KGF-2 antagonists may also be employed to treatglomerulonephritis, which is characterized by the marked proliferationof glomerular epithelial cells which form a cellular mass fillingBowman's space.

[0296] The antagonists may also be employed to inhibit theover-production of scar tissue seen in keloid formation after surgery,fibrosis after myocardial infarction or fibrotic lesions associated withpulmonary fibrosis and restenosis. KGF-2 antagonists may also beemployed to treat other proliferative diseases which are stimulated byKGF-2, including cancer and Kaposi's sarcoma.

[0297] KGF-2 antagonists may also be employed to treat keratitus whichis a chronic infiltration of the deep layers of the cornea with uvealinflammation characterized by epithelial cell proliferation.

[0298] The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

[0299] The polypeptides, agonists and antagonists of the presentinvention may be employed in combination with a suitable pharmaceuticalcarrier to comprise a pharmaceutical composition. Such compositionscomprise a therapeutically effective amount of the polypeptide, agonistor antagonist and a pharmaceutically acceptable carrier or excipient.Such a carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration.

[0300] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such containers can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides, agonists and antagonists of the presentinvention may be employed in conjunction with other therapeuticcompounds.

[0301] The polypeptide having KGF-2 activity may be administered inpharmaceutical compositions in combination with one or morepharmaceutically acceptable excipients. It will be understood that, whenadministered to a human patient, the total daily usage of thepharmaceutical compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the type and degree ofthe response to be achieved; the specific composition an other agent, ifany, employed; the age, body weight, general health, sex and diet if thepatient; the time of administration, route of administration, and rateof excretion of the composition; the duration of the treatment; drugs(such as a chemotherapeutic agent) used in combination or coincidentalwith the specific composition; and like factors well known in themedical arts. Suitable formulations, known in the art, can be found inRemington's Pharmaceutical Sciences (latest edition), Mack PublishingCompany, Easton, Pa.

[0302] The KGF-2 composition to be used in the therapy will beformulated and dosed in a fashion consistent with good medical practice,taking into account the clinical condition of the individual patient(especially the side effects of treatment with KGF-2 alone), the site ofdelivery of the KGF-2 composition, the method of administration, thescheduling of administration, and other factors known to practitioners.The “effective amount” of KGF-2 for purposes herein is thus determinedby such considerations.

[0303] The pharmaceutical compositions may be administered in aconvenient manner such as by the oral, topical, intravenous,intraperitoneal, intramuscular, intraarticular, subcutaneous,intranasal, intratracheal or intradermal routes. The pharmaceuticalcompositions are administered in an amount which is effective fortreating and/or prophylaxis of the specific indication. In most cases,the dosage is from about 1 μg/kg to about 30 mg/kg body weight daily,taking into account the routes of administration, symptoms, etc.However, the dosage can be as low as 0.001 μg/kg. For example, in thespecific case of topical administration dosages are preferablyadministered from about 0.01 μg to 9 mg per cm².

[0304] As a general proposition, the total pharmaceutically effectiveamount of the KGF-2 administered parenterally per more preferably dosewill be in the range of about 1 μg/kg/day to 100 mg/kg/day of patientbody weight, although, as noted above, this will be subject totherapeutic discretion. If given continuously, the KGF-2 is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution or bottle solution may also be employed.

[0305] A course of KGF-2 treatment to affect the fibrinolytic systemappears to be optimal if continued longer than a certain minimum numberof days, 7 days in the case of the mice. The length of treatment neededto observer changes and the interval following treatment for responsesto occur appears to vary depending on the desired effect. Such treatmentlengths are indicated in the Examples below.

[0306] The KGF-2 polypeptide is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U. Sidman etal, Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate)(R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R.Langer, Chem. Tech 12:98-105 (1982)), ethylene vinyl acetate (R. Langeret al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release KGF-2 compositions also include liposomally entrappedKGF-2. Liposomes containing KGF-2 are prepared by methods known per se:DE 3,218,121; Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034 (1980); EP52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal KGF-2 therapy.

[0307] For parenteral administration, in one embodiment, the, KGF-2 isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

[0308] Generally, the formulations are prepared by contacting the KGF-2uniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes. Suitable formulations, known in the art, can be found inRemington's Pharmaceutical Sciences (latest edition), Mack PublishingCompany, Easton, Pa.

[0309] The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

[0310] KGF-2 is typically formulated in such vehicles at a concentrationof about 0.01 μg/ml to 100 mg/ml, preferably 0.01 μg/ml to 10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of KGF-2 salts.

[0311] KGF-2to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic KGF-2compositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

[0312] KGF-2 ordinarily will be stored in unit or multi-dose containers,for example, sealed ampules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous KGF-2 solution, and the resultingmixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized KGF-2 using bacteriostaticWater-for-Injection.

[0313] Dosaging may also be arranged in a patient specific manner toprovide a predetermined concentration of an KGF-2 activity in the blood,as determined by an RIA technique, for instance. Thus patient dosagingmay be adjusted to achieve regular on-going trough blood levels, asmeasured by RIA, on the order of from 50 to 1000 ng/ml, preferably 150to 500 ng/ml.

[0314] Pharmaceutical compositions of the invention may be administeredorally, rectally, parenterally, intracisternally, intradermally,intravaginally, intraperitoneally, topically (as by powders, ointments,gels, creams, drops or transdermal patch), bucally, or as an oral ornasal spray. By “pharmaceutically acceptable carrier” is meant anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraaticular injection and infusion.

[0315] Preferred KGF-2 formulations are described in U.S. ProvisionalAppln. No. 60/068493, filed Dec. 22, 1997, which is herein incorporatedby reference.

[0316] The KGF-2 polypeptides, agonists and antagonists which arepolypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

[0317] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

[0318] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle. Examples of other delivery vehicles include an HSV-based vectorsystem, adeno-associated virus vectors, and inert vehicles, for example,dextran coated ferrite particles.

[0319] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoria Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0320] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller et al, Biotechniques Vol. 7, No. 9:980-990 (1989),or any other promoter (e.g., cellular promoters such as eukaryoticcellular promoters including, but not limited to, the histone, pol III,and β-actin promoters). Other viral promoters which may be employedinclude, but are not limited to, adenovirus promoters, thymidine kinase(TK) promoters, and B19 parvovirus promoters. The selection of asuitable promoter will be apparent to those skilled in the art from theteachings contained herein.

[0321] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0322] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cell lineswhich may be transfected include, but are not limited to, the PE501,PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy 1:5-14 (1990), which is incorporated herein by reference in itsentirety. The vector may transduce the packaging cells through any meansknown in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

[0323] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epitheliel cells.

[0324] Chromosome Assays

[0325] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0326] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0327] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0328] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal, Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, NewYork (1988).

[0329] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusilk, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0330] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0331] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0332] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0333] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0334] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0335] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0336] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D., et al., NucleicAcids Res., 8:4057 (1980).

[0337] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a frangment that has not beendephosphorylated.

[0338] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0339] A cell has been “transformed” by exogenous DNA when suchexogenous DNA has been introduced inside the cell membrane. ExogenousDNA may or may not be integrated (covalently linked) inter-chromosomalDNA making the genome of the cell. Prokaryote and yeast, for example,the exogenous DNA may be maintained on an episomal element, such aplasmid. With respect to eukaryotic cells, a stably transformed ortransfected cell is one in which the exogenous DNA has become integratedinto the chromosome so that it is inherited by daughter cells throughchromosome replication. This ability is demonstrated by the ability ofthe eukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cell containing the exogenouis DNA. An example oftransformation is exhibited in Graham, F. & Van der Eb, A., Virology,52:456457 (1973).

[0340] “Transduction” or “transduced” refers to a process by which cellstake up foreign DNA and integrate that foreign DNA into theirchromosome. Transduction can be accomplished, for example, bytransfection, which refers to various techniques by which cells take upDNA, or infection, by which viruses are used to transfer DNA into cells.

EXAMPLE 1

[0341] Bacterial Expression and Purification of KGF-2

[0342] The DNA sequence encoding KGF-2, ATCC # 75977, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ end sequences of the processed KGF-2 cDNA (including the signalpeptide sequence). The 5′ oligonucleotide primer has the sequence 5′CCCCACATGTGGAAATGGATACTGACACATTGTGCC 3′ (SEQ ID No. 3) contains an AflIII restriction enzyme site including and followed by 30 nucleotides ofKGF-2 coding sequence starting from the presumed initiation codon. The3′ sequence 5′ CCCAAGCTTCCACAAACGTTGCCTTCCTCTATGAG 3′ (SEQ ID No. 4)contains complementary sequences to Hind III site and is followed by 26nucleotides of KGF-2. The restriction enzyme sites are compatible withthe restriction enzyme sites on the bacterial expression vector pQE-60(Qiagen, Inc. Chatsworth, Calif.). pQE-60 encodes antibiotic resistance(Amp¹), a bacterial origin of replication (ori), an IPTG-regulatablepromoter operator (P/0), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-60 is then digested with NcoI and HindIII.The amplified sequences are ligated into pQE-60 and are inserted inframe. The ligation mixture is then used to transform E. coli strainM15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J., etal., Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989). M15/rep4 contains multiple copies of the plasmid pREP4,which expresses the lacI repressor and also confers kanamycin resistance(Kan¹). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies are selected. PlasmidDNA is isolated and confirmed by restriction analysis. Clones containingthe desired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG interacts with thelacI repressor to cause it to dissociate from the operator, forcing thepromoter to direct transcription. Cells are grown an extra 3 to 4 hours.Cells are then harvested by centrifugation. The cell pellet issolubilized in the chaotropic agent 6 Molar Guanidnine HCl. Afterclarification, solubilized KGF-2 is purified from this solution bychromatography on a Heparin affinity column under conditions that allowfor tight binding of the proteins (Hochuli, E., et al., J.Chromatography 411:177-184 (1984)). KGF-2 (75% pure) is eluted from thecolumn by high salt buffer.

EXAMPLE 2

[0343] Bacterial Expression and Purification of a Truncated Version ofKGF-2

[0344] The DNA sequence encoding KGF-2, ATCC # 75977, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ sequences of the truncated version of the KGF-2 polypeptide. Thetruncated version comprises the polypeptide minus the 36 amino acidsignal sequence, with a methionine and alanine residue being added justbefore the cysteine residue which comprises amino acid 37 of thefill-length protein. The 5′ oligonucleotide primer has the sequence 5′CATGCCATGGCGTGCCAAGCCCTTGGTCAGGACATG 3′ (SEQ ID No. 5) contains an NcoIrestriction enzyme site including and followed by 24 nucleotides ofKGF-2 coding sequence. The 3′ sequence 5′ CCCAAGCTTCCACAAACGTTGCCTTCCTCTATGAG 3′ (SE,Q ID No. 6) contains complementary sequences to Hind IIIsite and is followed by 26 nucleotides of the KGF-2 gene. Therestriction enzyme sites are compatible with the restriction enzymesites on the bacterial expression vector pQE-60 (Qiagen, Inc.Chatsworth, Calif.). pQE-60 encodes antibiotic resistance (Amp¹), abacterial origin of replication (ori), an IPTG-regulatable promoteroperator (P/0), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-60 is then digested with NcoI and HindIII.The amplified sequences are ligated into pQE-60 and are inserted inframe. The ligation mixture is then used to transform E. coli strainM15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J., etal., Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989). M15/rep4 contains multiple copies of the plasmid pREP4,which expresses the lacI repressor and also confers kanamycin resistance(Kang¹). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies are selected. PlasmidDNA is isolated and confirmed by restriction analysis. Clones containingthe desired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTS induces byinactivating the laci repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedKGF-2 is purified from this solution by chromatography on a Heparinaffinity column under conditions that allow for tight binding theproteins (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)).KGF-2 protein is eluted from the column by high salt buffer.

EXAMPLE 3

[0345] Cloning and Expression of KGF-2 Using the Baculovirus ExpressionSystem

[0346] The DNA sequence encoding the full length KGF-2 protein, ATCC #75977, is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene:

[0347] The 5′ primer has the sequence 5′GCGGGATCCGCCATCATGTGGAAATGGATACTCAC 3′ (SEQ ID No. 7) and contains aBamHI restriction enzyme site (in bold) followed by 6 nucleotidesresembling an efficient signal for the initiation of translation ineukaryotic cells (Kozak, M., J. Mol. Biol., 196:947-950 (1987)) and justbehind the first 17 nucleotides of the KGF-2 gene (the initiation codonor translation “ATG” is underlined).

[0348] The 3′ primer has the sequence 5′ GCGCGGTACCACAAACGTTGCCTTCCT 3′(SEQ ID No. 8) and contains the cleavage site for the restrictionendonuclease Asp718 and 19 nucleotides complementary to the 3′non-translated sequence of the KGF-2 gene. The amplified sequences areisolated from a 1% agarose gel using a commercially available kit fromQiagen, Inc., Chatsworth, Calif. The fragment is then digested with theendonucleases BamHI and Asp718 and then purified again on a 1% agarosegel. This fragment is designated F2.

[0349] The vector pA2 (modification of pVL941 vector, discussed below)is used for the expression of the KGF-2 protein using the baculovirusexpression system (for review see: Summers, M. D. & Smith, G. E., Amanual or methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555(1987)). This expression vector contains the strong polyhedrin promoterof the Autographa californica nuclear polyhidrosis virus (AcMNPV)followed by the recognition sites for the restriction endonucleasesBamHI and Asp718. The polyadenylation site of the simian virus (SV) 40is used for efficient polyadenylation. For an easy selection ofrecombinant viruses the beta-galactosidase gene from E. coli is insertedin the same orientation as the polyhedrin promoter followed by thepolyadenylation signal of the polyhedrin gene. The polyhedrin sequencesare flanked at both sides by viral sequences for the cell-mediatedhomologous recombination of co-transfected wild-type viral DNA. Manyother baculovirus vectors could be used such as pAc373, pVL941 andpAcIM1 (Luckow, V. A. & Summers, M. D., Virology, 170:31-39).

[0350] The plasmid is digested with the restriction enzymes BamHI andAsp718. The DNA is then isolated from a 1% agarose gel using thecommercially available kit (Qiagen, Inc., Chatsworth, Calif.). Thisvector DNA is designated V2.

[0351] Fragment F2 and the plasmid V2 are ligated with T4 DNA ligase. E.coli HB101 cells are then transformed and bacteria identified thatcontained the plasmid (pBacKGF-2) with the KGF-2 gene using PCR withboth cloning oligonucleotides. The sequence of the cloned fragment isconfirmed by DNA sequencing.

[0352] 5 μg of the plasmid pBacKGF-2 is co-transfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner, et al., Proc. Natl. Acad Sci. USA, 84:7413-7417 (1987)).

[0353] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacKGF-2are mixed in a sterile well of a microtiter plate containing 50 μl ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the tnansfectionmixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate is rocked back and forth to mix the newly addedsolution. The plate is then incubated for 5 hours at 27° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plate is put back into an incubator and cultivation continued at 27°C. for four days.

[0354] After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) is used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0355] Four days after the serial dilution, the viruses are added to thecells and blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses is then resuspendedin an Eppendorf tube containing 200 μl of Grace's medium. The agar isremoved by a brief centrifugation and the supernatant containing therecombinant baculovirus is used to infect Sf9 cells seeded in 35 mmdishes. Four days later the supernatants of these culture dishes areharvested and then stored at 4° C.

[0356] Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbzculovirus V-KGF-2 at a multiplicity of infection (MOI) of 2. Six hourslater the medium is removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 μCi of ³⁵S methionine and 5 μCi ³⁵S cysteine (Amersham) areadded. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 4

[0357] Most of the vectors used for the transient expression of theKGF-2 protein gene sequence in mammalian cells should carry the SV40origin of replication. This allows the replication of the vector to highcopy numbers in cells (e.g., COS cells) which express the T antigenrequired for the initiation of viral DNA synthesis. Any other mammaliancell line can also be utilized for this purpose.

[0358] A typical mammalian expression vector contains the promoterelement, which mediates the initiation of transcription of mRNA, theprotein coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV,HTLVI, HIVI and the immediate early promoter of the cytomegalovirus(CMV). However, cellular signals can also be used (e.g., human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela, 283, H9 and Jurkart cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV1, African green monkey cells, quail QC1-3cells, 293T cells, mouse L cells and Chinese hamster ovary cells.

[0359] Alternatively, the gene can be expressed in stable cell linesthat contain the gene integrated into a chromosome. The co-transfectionwith a selectable marker such as dhfr, gpt, neomycin, hygromycin allowsthe identification and isolation of the transfected cells.

[0360] The transfected gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase) is auseful marker to develop cell lines that carry several hundred or evenseveral thousand copies of the gene of interest. Another usefulselection marker is the enzyme glutamine synthase (GS) (Murphy et al.,Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology10:169-175 (1992)). Using these markers, the mammalian cells are grownin selective medium and the cells with the highest resistance areselected. These cell lines contain the amplified gene(s) integrated intoa chromosome. Chinese hamster ovary (CHO) cells are often used for theproduction of proteins.

[0361] The expression vectors pC1 and pC4 contain the strong promoter(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and CellularBiology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer(Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g.,with the restriction enzyme cleavage sites BamHI, XbaI and Asp718,facilitate the cloning of the gene of interest. The vectors contain inaddition the 3′ intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.

[0362] A. Expression of Recombinant KGF-2 in COS Cells

[0363] The expression of plasmid, KGF-2 HA was derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E. coli replication origin, 4) CMVpromoter followed by a polylinker region, a SV40 intron andpolyadenylation site. The HA tag correspond to an epitope derived fromthe influenza hemagglutinin protein as previously described (Wilson, I.,et al., Cell 37:767, (1984)). The infusion of HA tag to the targetprotein allows easy detection of the recombinant protein with anantibody that recognizes the HA epitope. A DNA fragment encoding theentire KGF-2 precursor HA tag fused in frame with the HA tag, therefore,the recombinant protein expression is directed under the CMV promoter.

[0364] The plasmid construction strategy is described as follows:

[0365] The DNA sequence encoding KGF-2, ATCC #75977, is constructed byPCR using two primers: the 5′ primer 5′TAACGAGGATCCGCCATCATGTGGAAATGGATACTGACACAC 3′ (SEQ ID No. 9) contains aBamHI site followed by 22 nucleotides of KGF-2 coding sequence startingfrom the initiation codon; the 3′ sequence 5′TAAGCACTCGAGTGAGTGTACCACCATTGGAAGAAATG 3′ (SEQ ID No. 10) containscomplementary sequences to an XhoI site, HA tag and the last 26nucleotides of the KGF-2 coding sequence (not including the stop codon).Therefore, the PCR product contains a BamHI site, KGF-2 coding sequencefollowed by an XhoI site, an HA tag fused in frame, and a translationtermination stop codon next to the HA tag. The PCR amplified DNAfragment and the vector, pcDNA-3HA, are digested with BamHI and XhoIrestriction enzyme and ligated resulting in pcDNA-3′HA-KGF-2. Theligation mixture is transformed into E. coli strain XL1 Blue (StratageneCloning Systems, La Jolla, Calif.) the transformed culture is plated onampicillin media plates and resistant colonies are selected. Plasmid DNAwas isolated from transformants and examined by PCR and restrictionanalysis for the presence of the correct fragment. For expression of therecombinant KGF-2, COS cells were transfected with the expression vectorby DEAE-DEXTRAN method (Sambrook, J., et al, Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory Press, (1989)). The expressionof the KGF-2 HA protein was detected by radiolabelling andimmunoprecipitation method (Harlow, E. & Lane, D., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cellswere labelled for 8 hours with ³⁵S-cysteine two days post transfection.Culture media were then collected and cells were lysed with detergent(RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 5 OmMTris, pH 7.5) (Wilson, I., et al., Id. 37:767 (1984)). Both cell lysateand culture media were precipitated with a HA specific monoclonalantibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.

[0366] B: Expression and Purification of Human KGF-2 Protein Using theCHO Expression System

[0367] The vector pC1 is used for the expression of KFG-2 protein.Plasmid pC1 is a derivative of the plasmid pSV2-dhfr [ATCC Accession No.37146]. Both plasmids contain the mouse DHFR gene under control of theSV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,Life Technologies) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W.,Kellems, R. M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta,1097:107-143, Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol.9:64-68). Cells grown in increasing concentrations of MTX developresistance to the drug by overproducing the target enzyme, DHFR, as aresult of amplification of the DHFR gene. If a second gene is linked tothe DHFR gene it is usually co-amplified and over-expressed. It is stateof the art to develop cell lines carrying more than 1,000 copies of thegenes. Subsequently, when the methotrexate is withdrawn, cell linescontain the amplified gene integrated into the chromosome(s).

[0368] Plasmid pC1 contains for the expression of the gene of interest astrong promoter of the long terminal repeat (LTR) of the Rouse SarcomaVirus (Cullen, et al., Molecular and Cellular Biology, March1985:438-4470) plus a fragment isolated from the enhancer of theimmediate early gene of human cytomegalovirus (CMV) (Boshart et al.,Cell 41:521-530, 1985). Downstream of the promoter are the followingsingle restriction enzyme cleavage sites that allow the integration ofthe genes: BamHI, Pvull, and Nrul. Behind these cloning sites theplasmid contains translational stop codons in all three reading framesfollowed by the 3′ intron and the polyadenylation site of the ratpreproinsulin gene. Other high efficient promoters can also be used forthe expression, e.g., the human β-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g.,from the human growth hormone or globin genes can be used as well.

[0369] Stable cell lines carrying a gene of interest integrated into thechromosomes can also be selected upon co-transfection with a selectablemarker such as gpt, G418 or hygromycin. It is advantageous to use morethan one selectable marker in the beginning, e.g., G418 plusmethotrexate.

[0370] The plasmid pC1 is digested with the restriction enzyme BamHI andthen dephosphorylated using calf intestinal phosphates by proceduresknown in the art. The vector is then isolated from a 1% agarose gel.

[0371] The DNA sequence encoding KFG-2, ATCC No. 75977, is amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the gene:

[0372] The 5′ primer has the sequence 5′TAACGAGGATCCGCCATCATGTGGAAATGGATACTGACAC 3′ (SEQ ID No. 9) containingthe underlined BamHI restriction enzyme site followed by 21 bases of thesequence of KGF-2 of FIG. 1 (SEQ ID NO: 1). Inserted into an expressionvector, as described below, the 5′ end of the amplified fragmentencoding human KGF-2 provides an efficient signal peptide. An efficientsignal for initiation of translation in eukaryotic cells, as describedby Kozak M., J. Mol. Biol. 196:947-950 (1987) is appropriately locatedin the vector portion of the construct.

[0373] The 3′ primer has the sequence 5′TAAGCAGGATCCTGAGTGTACCACCATTGGAAGAAATG 3′ (SEQ ID NO. 10) containing theBamHI restriction followed by nucleotides complementary to the last 26nucleotides of the KGF-2 coding sequence set out in FIG. 1 (SEQ ID NO:1), not including the stop codon.

[0374] The amplified fragments are isolated from a 1% agarose gel asdescribed above and then digested with the endonuclease BamHI and thenpurified again on a 1% agarose gel.

[0375] The isolated fragment and the dephosphorylated vector are thenligated with T4 DNA ligase. E. coli HB101 cells are then transformed andbacteria identified that contained the plasmid pC1. The sequence andorientation of the inserted gene is confirmed by DNA sequencing.

[0376] Transfection of CHO-DHFR-cells

[0377] Chinese hamster ovary cells lacking an active DHFR enzyme areused for transfection. 5 μg of the expression plasmid C1 arecotransfected pith 0.5 μg of the plasmid pSVneo using the lipofectingmethod (Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the gene neo from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) and cultivated for 10-14 days. After this period,single clones are trypsinized and then seeded in 6-well petri dishesusing different concentrations of methotrexate (25 nM, 50 nM, 100 nM,200 nM, 400 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (500 nM, 1 μM, 2 μM, 5 μM). Thesame procedure is repeated until clones grow at a concentration of 100μM.

[0378] The expression of the desired gene product is analyzed by Westernblot analysis and SDS-PAGE.

EXAMPLE 5

[0379] Transcription and Translation of Recombinant KGF-2 in vitro

[0380] A PCR product is derived from the cloned cDNA in the pA2 vectorused for insect cell expression of KGF-2. The primers used for this PCRwere: 5′ ATTAACCCTCACTAAAGGGAGGCCATGTGGAAATGGATACTGACA CATTGTGCC 3′ (SEQID No. 11) and 5′CCCAAGCTTCCACAAACGTTGCCTTCCTCTATGAG 3′(SEQ ID No. 12).

[0381] The first primer contains the sequence of a T3 promoter 5′ to theATG initiation codon. The second primer is complimentary to the 3′ endof the KGF-2 open reading frame, and encodes the reverse complement of astop codon.

[0382] The resulting PCR product is purified using a commerciallyavailable kit from Qiagen. 0.5 μg of this DNA is used as a template foran in vitro transcription-translation reaction. The reaction isperformed with a kit commercially available from Promega under the nameof TNT. The assay is performed as described in the instructions for thekit, using radioactively labeled methionine as a substrate, with theexception that only {fraction (1/2)} of the indicated volumes ofreagents are used and that the reaction is allowed to proceed at 33° C.for 1.5 hours.

[0383] Five μl of the reaction is electrophoretically separated on adenaturing 10 to 15% polyacrylamide gel. The gel is fixed for 30 minutesin a mixture of water:Methanol:Acetic acid at 6:3:1 volumesrespectively. The gel is then dried under heat and vacuum andsubsequently exposed to an X-ray film for 16 hours. The film isdeveloped showing the presence of a radioactive protein bandcorresponding in size to the conceptually translated KGF-2, stronglysuggesting that the cloned cDNA for KGF-2 contains an open reading framethat codes for a protein of the expected size.

EXAMPLE 6

[0384] Expression via Gene Therapy

[0385] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added.) This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0386] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988)) flanked bythe long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0387] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an EcoRI site and the3′ primer further includes a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the amplified EcoRI andHindIII fragment are added together, in the presence of T4 DNA ligase.The resulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

[0388] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0389] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and isquickly replaced with the media from the producer cells. This media isremoved and replaced with fresh media. If the titer of virus is high,then virtually all fibroblasts will be infected and no selection isrequired. If the titer is very low, then it is necessary to use aretroviral vector that has a selectable marker, such as neo or his.

[0390] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

EXAMPLE 7

[0391] KGF-2 Stimulated Wound Healing in the Diabetic Mouse Model

[0392] To demonstrate that keratinocyte growth factor-2 (KGF-2) wouldaccelerate the healing process, the genetically diabetic mouse model ofwound healing was used. The full thickness wound healing model in thedb+/db+ mouse is a well characterized, clinically relevant andreproducible model of impaired wound healing. Healing of the diabeticwound is dependent on formation of granulation tissue andre-epithelialization rather than contraction (Gartner, M. H. et al.,J.Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol.136:1235 (1990)).

[0393] The diabetic animals have many of the characteristic featuresobserved in Type II diabetes mellitus. Homozygous (db+/db+) mice areobese in comparison to their normal heterozygous (db+/+m) littermates.Mutant diabetic (db+/db+) mice have a single autosomal recessivemutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci.USA 77:283-293 (1982)). Animals show polyphagia, polydipsia andpolyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose,increased or normal insulin levels, and suppressed cell-mediatedimmunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M.et al., Clin. Exp. Immunol. 51(1): 1-7 (1983); Leiter et al., Am. J. ofPathol. 114:46-55 (1985)). Peripheral neuropathy, myocardialcomplications, and microvascular lesions, basement membrane thickeningand glomeruldar filtration abnormalities have been described in theseanimals (Norido, F. et al., Exp. Neurol. 83(2): 221-232 (1984);Robertson et al., Diabetes 29(1):60-67 (1980); Giacomelli et al., LabInvest. 40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6(1982)). These homozygous diabetic mice develop hyperglycemia that isresistant to insulin analogous to human type II diabetes (Mandel et al.,J. Immunol. 120:1375-1377 (1978)).

[0394] The characteristics observed in these animals suggests thathealing in this model may be similar to the healing observed in humandiabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).The results of this study demonstrated that KGF-2 has a potentstimulatory effect on the healing of full thickness wounds in diabeticand nondiabetic heterozygous littermates. Marked effects onre-epithelialization and an increase in collagen fibrils, granulationtissue within the dermis were observed in KGF-2 treated animals. Theexogenous application of growth factors may accelerate granulationtissue formation by drawing inflammatory cells into the wound.

[0395] Animals

[0396] Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates were used in this study(Jackson Laboratories). The animals were purchased at 6 weeks of age andwere 8 weeks old at the beginning of the study. Animals wereindividually housed and received food and water ad libitum. Allmanipulations were performed using aseptic techniques. The experimentswere conducted according to tie rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

[0397] KGF-2

[0398] The recombinant human KGF-2 used for the wound healing studieswas over-expressed, and purified from pQE60-Cys37, an E. coli expressionvector system (pQE-9, Qiagen). The protein expressed from this constructis the KGF-2 from Cystein at position 37 to Serine at position 208 witha 6X(His) tag attached to the N-terminus of the protein (SEQ ID NOS:29-30) (FIG. 15). Fractions containing greater than 95% pure recombinantmaterials were used for the experiment. Keratinocyte growth factor-2 wasformulated in a vehicle containing 100 mM Tris, 8.0 and 600 mM NaCl. Thefinal concentrations were 80 μg/mL and 8 μg/mL of stock solution.Dilutions were made from stock solution using the same vehicle.

[0399] Surgical Wounding

[0400] Wounding protocol was performed according to previously reportedmethods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245.251 (1990)).Briefly, on the day of wounding, animals were anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal was shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area was dried with sterile gauze prior towounding. An 8 mm full-thickness wound was then created using a Keyestissue punch. Immediately following wounding, the surrounding skin wasgently stretched to eliminate wound expansion. The wounds were left openfor the duration of the experiment. Application of the treatment wasgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds were gently cleansed with sterilesaline and gauze sponges.

[0401] Wounds were visually examined and photographed at a fixeddistance at the day of surgery and at two day intervals thereafter.Wound closure was determined by daily measurement on days 1-5 and on day8. Wounds were measured horizontally and vertically using a calibratedJameson caliper. Wounds were considered healed if granulation tissue wasno longer visible and the wound was covered by a continuous epithelium.

[0402] KGF-2 was administered using two different doses of KGF-2, one at4 μg per wound per day for 8 days and the second at 40 μg per wound perday for 8 days in 50 μL of vehicle. Vehicle control groups received 50μL of vehicle solution.

[0403] Animals were euthanized on day 8 with an intraperitonealinjection of sodium pentobarbital (300 mg/kg). The wounds andsurrounding skin were then harvested for histology andimmunohistochemistry. Tissue specimens were placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

[0404] Experimental Design

[0405] Three groups of 10 animals each (5 diabetic and 5 non-diabieticcontrols) were evaluated: 1) Vehicle placebo control, 2) KGF-2 4 μg/dayand 3) KGF-2 40 μg/day. This study was designed as follows: N GroupTreatment N = 5 db+/db+ vehicle 50 μL N = 5 db+/+m vehicle 50 μL N = 5db+/db+ KGF-2 4 μg/50 μL N = 5 db+/+m KGF-2 4 μg/50 μL N = 5 db+/db+KGF-2 40 μg/50 μL N = 5 db+/+m KGF-2 40 μg/50 μL

[0406] Measurement of Wound Area and Closure

[0407] Wound closure was analyzed by measuring the area in the verticaland horizontal axis and obtaining the total square area of the wound.Contraction was then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 was; 64 mm², the corresponding size of the dermalpunch. Calculations were made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[0408] Histology

[0409] Specimens were fixed in 10% buffered formalin and paraffinembedded blocks were sectioned perpendicular to the wound surface (5 μm)and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining was performed on cross-sections of bisected wounds. Histologicexamination of the wounds were used to assess whether the healingprocess and the morphologic appearance of the repaired skin was alteredby treatment with KGF-2. This assessment included verification of thepresence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)) (Table 1). A calibrated lensmicrometer was used by a blinded observer.

[0410] Immunohistochemistry

[0411] Re-epithelialization

[0412] Tissue sections were stained immunohistochemically with apolyclonal rabbit anti-human keratin antibody using ABC Elite detectionsystem. Human skin was used as a positive tissue control whilenon-immune IgG was used as a negative control. Keratinocyte growth wasdetermined by evaluating the extent of reepithelialization of the woundusing a calibrated lens micrometer.

[0413] Cell Proliferation Marker

[0414] Proliferating cell nuclear antigen/cyclin (PCNA) in skinspecimens was demonstrated by using anti-PCNA antibody (1:50) with anABC Elite detection system. Human colon cancer served as a positivetissue control and human brain tissue was used as a negative tissuecontrol. Each specimen included a section with omission of the primaryantibody and substitution with non-immune mouse IgG. Ranking of thesesections was based on the extent of proliferation on a scale of 0-8, thelower side of the scale reflecting slight proliferation to th

[0415] e higher side reflecting intense proliferation.

[0416] Statistical Analysis

[0417] Experimental data were analyzed using an unpaired t test. A pvalue of <0.05 was considered significant. The data were expressed asthe mean±SEM.

[0418] Results

[0419] Effect of KGF-2 on Wound Closure

[0420] Diabetic mice showed impaired healing compared to heterozygousnormal mice. The dose of 4 μg of KGF-2 per site appeared to producemaximum response in diabetic and non-diabetic animals (FIG. 5, 6). Theseresults were statistically significant (p=0.002 and p<0.0001) whencompared with the buffer control groups. Treatment with KGF-2 resultedin a final average closure of 60.6% in the group receiving 4 μg/day and34.5% in the 40 μg/day group. Wounds in the buffer control group hadonly 3.8% closure by day 8. Repeated measurements of wounds on days 2-5post-wounding and on day 8 taken from the db+/db+ mice treated withKGF-2 demonstrated a significant improvement in the total wound area(sq. mm) by day 3 post-wounding when compared to the buffer controlgroup. This improvement continued and by the end of the experiment,statistically significant results were observed (FIG. 7). Animals in thedb/+m groups receiving KGF-2 also showed a greater reduction in thewound area compared to the buffer control groups in repetitivemeasurements (FIG. 8). These results confirmed a greater rate of woundclosure in the KGF-2 treated animals.

[0421] Effect of KGF-2 on Histologic Score

[0422] Histopathologic evaluation of KGF-2 in the diabetic (db+/db+)model on day 8 demonstrated a statistically significant improvement(p<0.00001) in the wound score when compared with the buffer control.The pharmacologic effects observed with both the 4 μg and the 40 μgdoses of KGF-2 were not significantly different from each other. Thebuffer control group showed minimal cell accumulation with nogranulation tissue or epithelial travel while the 4 μg and 40 μg dosesof KGF-2 (p<0.0001 & p=0.06 respectively) displayed epithelium coveringthe wound, neovascularization, granulation tissue formation andfibroblast and collagen deposition (FIG. 9).

[0423] Histopathologic assessment of skin wounds was performed onhematoxylin-eosin stained samples. Scoring criteria included a scale of1-12, a score of one representing minimal cell accumulation with littleto no granulation and a score of 12 representing the abundant presenceof fibroblasts, collagen deposition and new epithelium covering thewound (Table 1). TABLE 1 Scoring of Histology Sections Score Criteria1-3 None to minimal cell accumulation. No granulation tissue orepithelial travel. 4-6 Thin, immature granulation that is dominated byinflammatory cells but has few fibroblasts, capillaries or collagendeposition. Minimal epithelial migration. 7-9 Moderately thickgranulation tissue, can range from being dominated by inflammatory cellsto more fibroblasts and colla- gen deposition. Extensiveneovascularization. Epithelium can range from minimal to moderatemigration. 10-12 Thick, vascular granulation tissue dominated byfibroblasts and extensive collagen deposition. Epithelium partially tocompletely covering the wound.

[0424] Evaluation of the non diabetic littermates, after both doses ofKGF-2, showed no significant activity in comparison with the buffercontrol group for all measurements evaluated (FIG. 10). The buffercontrol group showed immature granulation tissue, inflammatory cells,and capillaries. The mean score was higher than the diabetic groupindicating impaired healing in the diabetic (db+/db+) mice.

[0425] Effect of KGF-2 on Re-epithelialization

[0426] Cytokeratine Immunostaining was used to determine the extent ofre-epithelialization. Scores were given based on degree of closure on ascale of 0 (no closure) to 8 (complete closure). In the groups receiving4 μg/day, there was a statistically significant improvement on there-epithelialization a score when compared to the buffer control groupp<0.001 (FIG. 11). In this group, keratinocytes were observed localizedin the newly formed epidermis covering the wound. Both doses of KGF-2also exhibited mitotic figures in various stages. Assessment of thenon-diabetic groups at both doses of KGF-2 also significantly improvedreepithelialization ranking (p=0.006 and 0.01 respectively,) (FIG. 12).

[0427] Effect of KGF-2 on Cell Proliferation

[0428] Proliferating cell nuclear antigen immunostaining demonstratedsignificant proliferation in both the 4 μg and 40 μg groups (FIG. 13).The non-diabetic group displayed similar results as both groupsreceiving both doses of KGF-2 showed higher significant scoring comparedto the buffer control group (FIG. 14). Epidermal proliferation wasobserved especially on the basal layer of the epidermis. In addition,high density PCNA-labeled cells were observed in the dermis, especiallyin the hair follicles.

[0429] Conclusion

[0430] The results demonstrate that KGF-2 specifically stimulates growthof primary epidermal keratinocytes. In addition, these experimentsdemonstrate that topically applied recombinant human KGF-2 markedlyaccelerates the rate of healing of full-thickness excisional dermalwounds in diabetic mice. Histologic assessment shows KGF-2 to inducekeratinocyte proliferation with epidermal thickening. This proliferationis localized in the basal layer of the epidermis as demonstrated byproliferating cell nuclear antigen (PCNA). At the level of the dermis,collagen deposition, fibroblast proliferation, and neo-vascularizationre-established the normal architecture of the skin.

[0431] The high density of PCNA-labeled cells on KGF-2 treated animalsin contrast with the buffer group, which had fewer PCNA-labeled cells,indicates the stimulation of keratinocytes at the dermal-epidermallevel, fibroblasts and hair follicles. The enhancement of the healingprocess by KGF-2 was consistently observed in this experiment. Thiseffect was statistically significant in the parameters evaluated(percent re-epithelialization and wound closure). Importantly,PCNA-labeled keratinocytes were mainly observed at the lower-basal layerof the epidermis. The dermis showed normalized tissue with fibroblasts,collagen, and granulation tissue.

[0432] The activity observed in the non-diabetic animals indicates thatKGF-2 had significant pharmacologic response in the percentage of woundclosure at day 8, as well as during the course of the experiment, basedon daily measurements. Although the histopathologic evaluation was notsignificantly different when compared with the buffer control,keratinocyte growth and PCNA scores demonstrated significant effects.

[0433] In summary, these results demonstrated that KGF-2 showssignificant activity in both impaired and normal excisional wound modelsusing the db+/+ mouse model and therefore may be useful in the treatmentof wounds including surgical wounds, diabetic ulcers, venous stasisulcers, burns, and other skin conditions.

Example 8

[0434] KGF-2 Mediated Wound Healing in the Steroid-Impaired Rat Model

[0435] The inhibition of wound healing by steroids has been welldocumented in various in vitro and in vivo systems (Wahl, S. M.Glucocorticoids and Wound healing. In Anti-Inflammatory Steroid Action:Basic and Clinical Aspects. 280-302 (1989); Wahl, S. M. et al., J.Immunol. 115: 476-481 (1975); Werb, Z. et al., J. Exp. Med.147:1684-1694 (1978)). Glucocorticoids retard wound healing byinhibiting angiogenesis, decreasing vascular permeability ( Ebert, R.H., et al., An. Intern. Med. 37:701-705 (1952)), fibroblastproliferation, and collagen synthesis (Beck, L. S. et al., GrowthFactors. 5: 295-304 (1991); Haynes, B. F., et al., J. Clin. Invest. 61:703-797 (1978)) and producing a transient reduction of circulatingmonocytes (Haynes, B. F., et al., J. Clin. Invest. 61: 703-797 (1978);Wahl, S. M. Glucocorticoids and wound healing. In AntiinflammatorySteroid Action: Basic and Clinical Aspects. Academic Press. New York.pp. 280-302 (1989)). The systemic administration of steroids to impairedwound healing is a well establish phenomenon in rats (Beck, L. S. etal., Growth Factors. 5: 295-304 (1991); Haynes, B. F., et al., J. Clin.Invest. 61: 703-797 (1978); Wahl, S. M. Glucocorticoids and woundhealing. In Antiinflammatory Steroid Action: Basic and Clinical Aspects.Academic Press. New York. pp. 280-302 (1989); Pierce, G. F., et al.,Proc. Natl. Acad. Sci. USA. 86: 2229-2233 (1989)).

[0436] To demonstrate that KGF-2 would accelerate the healing process,the effects of multiple topical applications of KGF-2 on full thicknessexcisional skin wounds in rats in which healing has been impaired by thesystemic administration of methylprednisolone was assesed. In vitrostudies have demonstrated that KGF-2 specifically stimulates growth ofprimary human epidermal keratinocytes. This example demonstrates thattopically applied recombinant human KGF-2 accelerates the rate ofhealing of full-thickness excisional skin wounds in rats by measuringthe wound gap with a calibrated Jameson caliper and by histomorphometryand immunohistochemistry. Histologic assessment demonstrates that KGF-2accelerates re-epithelialization and subsequently, wound repair.

[0437] Animals

[0438] Young adult male Sprague Dawley rats weighing 250-300g (CharlesRiver Laboratories) were used in this example. The animals werepurchased at 8 weeks of age and were 9 weeks old at the beginning of thestudy. The healing response of rats was impaired by the systemicadministration of methylprednisolone (17 mg/kg/rat intramuscularly) atthe time of wounding. Animals were individually housed and received foodand water ad libitum. All manipulations were performed using aseptictechniques. This study was conducted according to the rules andguidelines of Human Genome Sciences, Inc. Institutional Animal Care andUse Committee and the Guidelines for the Care and Use of LaboratoryAnimals.

[0439] KGF-2

[0440] Recombinant human KGF-2 was over-expressed and purified frompQE60-Cys37, an E. coli expression vector system (pQE-9, Qiagen). Theprotein expressed from this construct is the KGF-2 from Cysteine atposition 37 to Serine at position 208 with a 6X(His) tag attached to theN-terminus of the protein (FIG. 15) (SEQ ID NOS: 29-30). Fractionscontaining greater than 95% pure recombinant materials were used for theexperiment. KGF-2 was formulated in a vehicle containing 1×PBS. Thefinal concentrations were 20 μg/mL and 80 μg/mL of stock solution.Dilutions were made from stock solution using the same vehicle.

[0441] KGF-2Δ28 was over-expressed and purified from an E. coliexpression vector system. Fractions containing greater than 95% purerecombinant materials were used for the experiment. KGF-2 was formulatedin a vehicle containing 1×PBS. The final concentrations were 20 μg/mLand 80 μg/mL of stock solution. Dilutions were made from stock solutionusing the same vehicle.

[0442] Surgical Wounding

[0443] The wounding protocol was followed according to Example 7, above.On the day of wounding, animals were anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal was shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area was dried with sterile gauze priorto wounding. An 8 mm full-thickness wound was created using a Keyestissue punch. The wounds were left open for the duration of theexperiment. Applications of the testing materials were given topicallyonce a day for 7 consecutive days commencing on the day of wounding andsubsequent to methylprednisolone administration. Prior to treatment,wounds were gently cleansed with sterile saline and gauze sponges.

[0444] Wounds were visually examined and photographed at a fixeddistance at the day of wounding and at the end of treatment. Woundclosure Was determined by daily measurement on days 1-5 and on day 8 forFigure. Wounds were measured horizontally and vertically using acalibrated Jameson caliper. Wounds were considered healed if granulationtissue was no longer visible and the wound was covered by a continuousepithelium.

[0445] A dose response was performed using two different doses of KGF-2,one at 1 μg per wound per day and the second at 4 μg per wound per dayfor 5 days in 50 μL of vehicle. Vehicle control groups received 50 μL of1×PB.

[0446] Animals were euthanized on day 8 with an intraperitonealinjection of sodium pentobarbital (300 mg/kg). The wounds andsurrounding skin were then harvested for histology. Tissue specimenswere placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

[0447] Experimental Design

[0448] Four groups of 10 animals each (5 with methylprednisolone and 5without glucocorticoid) were evaluated: 1) Untreated group 2) Vehicleplacebo control 3) KGF-2 μg/day and 4) KGF-2 4 μg/day. This study wasdesigned as follows: n Group Treatment Glucocorticoid-Treated N = 5Untreated — N = 5 Vehicle 50 μL N = 5 KGF-2 (1 μg) 50 μL N = 5 KGF-2 (4μg) 50 μL Without Glucocorticoid N = 5 Untreated — N = 5 Vehicle 50 μL N= 5 KGF-2 (1 μg) 50 μL N = 5 KGF-2 (4 μg) 50 μL

[0449] Measurement of Wound Area and Closure

[0450] Wound closure was analyzed by measuring the area in the verticaland horizontal axis and obtaining the total area of the wound. Closurewas then estimated by establishing the differences between the initialwound area (day 0) and that of post treatment (day 8). The wound area onday 1 was 64 mm², the corresponding size of the dermal punch.Calculations were made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

[0451] Histology

[0452] Specimens were fixed in 10% buffered formalin and paraffinembedded blocks were sectioned perpendicular to the wound surface (5 μm)and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E)staining was performed on cross-sections of bisected wounds. Histologicexamination of the wounds allowed us to assess whether the healingprocess and the morphologic appearance of the repaired skin was improvedby treatment with KGF-2. A calibrated lens micrometer was used by ablinded observer to determine the distance of the wound gap.

[0453] Statistical Analysis

[0454] Experimental data were analyzed using an unpaired t test. A pvalue of <0.05 was considered significant. The data was expressed as themean±SEM.

[0455] Results

[0456] A comparison of the wound closure of the untreated control groupswith and without methylprednisolone demonstrates thatmethylprednisiolone-treated rats have significant impairment of woundhealing at 8 days post-wounding compared with normal rats. The totalwound area measured 58.4 mm² in the methylprednisolone injected groupand 22.4 mm² in the group not receiving glucocorticoid (FIG. 16).

[0457] Effect of KGF-2 on Wound Closure

[0458] Systemic administration of methylprednisolone in rats at the timeof wounding delayed wound closure (p=0.002) of normal rats. Woundclosure measurements of the methlyprednisolone-impaired groups at theend of the experiment on day 8 demonstrated that wound closure withKGF-2 was significantly greater statistically (1 μg p=0.002 & 4 μgp=0.005) when compared with the untreated group (FIG. 16). Percentagewound closure was 60.2% in the group receiving 1 μg KGF-2 (p=0.002) and73% in the group receiving 4 μg KGF-2 (p=0.0008). In contrast, the woundclosure of untreated group was 12.5% and the vehicle placebo group was28.6% (FIG. 17).

[0459] Longitudinal analysis of wound closure in the glucocorticoidgroups from day 1 to 8 shows a significant reduction of wound size fromday 3 to 8 postwounding in both doses of KGF-2 in the treated groups(FIG. 18).

[0460] The results demonstrate that the group treated with the 4 μgKGF-2 had statistically significant (p=0.05) accelerated wound closurecompared with the untreated group (FIG. 19A). Although it is difficultto assess the ability of a protein or other compounds to acceleratewound healing in normal animal (due to rapid recovery), nonetheless,KGF-2 was shown to accelerate wound healing in this model.

[0461] Histopathologic Evaluation of KGF-2 Treated Wounds

[0462] Histomorphometry of the wound gap indicated a reduction in thewound distance of the KGF-2 treated group. The wound gap observed forthe untreated group was 5336μ while the group treated with 1 μg KGF-2had a wound gap reduction to 29721μ; and the group treated with 4 μgKGF-2 (p=0.04) had a wound gap reduction to 3086μ. (FIG. 20)

[0463] Effects of KGF-2Δ28 in Wound Healing

[0464] Evaluation of KGF-2Δ28 and PDGF-BB in wound healing in themethylprednisolone impared rat model was also examined. The experimentwas carried out the same as for the KGF-2 protein above, except that theKGF-2Δ28 protein is not His tagged and wound healing was measured ondays 2, 4, 6, 8, and 10. The buffer vehicle for the proteins was 40 mMNaOAc and 150 mM NaCl, pH6.5 for all but the “E2” preparation of thefull length KGF-2. The buffer vehicle for the “E2” KGF-2 preparation was20 mM NaOAc and 4 mM NaCl, pH6.4.

[0465] The results shown in FIG. 19B demonstrate that KGF-2Δ28 hasstatistically significant accelerated wound closure compared with theuntreated group and has reversed the effects of methylprednisolone onwound healing.

[0466] Conclusions

[0467] This example demonstrates that KGF-2 reversed the effects ofmethylprednisolone on wound healing. The exogenous application of growthfactors may accelerate granulation tissue formation by drawinginflammatory cells into the wound. Similar activity was also observed inanimals not receiving methylprednisolone indicating that KGF-2 hadsignificant pharmacologic response in the percentage of wound closure atday 5 based on daily measurements. The glucocorticoid-impaired woundhealing model in rats was shown to be a suitable and reproducible modelfor measuring efficacy of KGF-2 and other compounds in the wound healingarea.

[0468] In summary, the results demonstrate that KGF-2 shows significantactivity in both glucocorticoid impaired and in normal excisional woundmodels. Therefore, KGF-2 may be clinically useful in stimulating woundhealing including surgical wounds, diabetic ulcers, venous stasisulcers, burns, and other abnormal wound healing conditions such asuremia, malnutrition, vitamin deficiencies and systemic treatment withsteroids and antineoplastic drugs.

EXAMPLE 9

[0469] Tissue Distribution of KGF-2 mRNA Expression

[0470] Northern blot analysis is carried out to examine the levels ofexpression of the gene encoding the KGF-2 protein in human tissues,using methods described by, among others, Sambrook et al., cited above,A probe corresponding to the entire open reading frame of KGF-2 of thepresent invention (SEQ ID NO: 1) was obtained by PCR and was labeledwith ³²P using the rediprime™ DNA labeling system (Amersham LifeScience), according to manufacturer's instructions. After labelling, theprobe was purified using a CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labelled probe was then used to examine varioushuman tissues for the expression of the gene encoding KGF-2.

[0471] Multiple Tissue Northern (MTN) blots containing poly A RNA fromvarious human tissues (H) or human immune system tissues (IM) wereobtained from Clontech and were examined with labelled probe usingExpressHyb™ Hybridization Solution (Clontech) according tomanufacturer's protocol number PT1190-1. Following hybridization andwashing, the blots are mounted and exposed to film at −70° C. overnight,and films developed according to standard procedures.

[0472] A major mRNA species of apporximately 4.2 kb was observed in mosthuman tissues. The KGF-2 mRNA was relatively abundant in heart,pancreas, placenta and ovary. A minor mRNA species of about 5.2 kb wasalso observed ubiquitously. The identity of this 5.2 kb mRNA species wasnot clear. It is possible that the 5.2 kb transcript encodes analternatively spliced form of KGF-2 or a third member of the KGF family.The KGF-2 cDNA was 4.1 kb, consistent with the size of the mRNA of 4.2kb.

EXAMPLE 10

[0473] Keratinocyte Proliferation Assays

[0474] Dermal keratinocytes are cells in the epidermis of the skin. Thegrowth and spreading of keratinocytes in the skin is an importantprocess in wound healing. A proliferation assay of keratinocyte istherefore a valuable indicator of protein activities in stimulatingkeratinocyte growth and consequently, wound healing.

[0475] Keratinocytes are, however, difficult to grow in vitro. Fewkeratinocyte cell lines exist. These cell lines have different cellularand genetic defects. In order to avoid complications of this assay bycellular defects such as loss of key growth factor receptors ordependence of key growth factors for growth, primary dermalkeratinocytes are chosen for this assay. These primary keratinocytes areobtained from Clonetics, Inc. (San Diego, Calif.).

[0476] Keratinocyte proliferation assay with alamarBlue

[0477] alamarBlue is a viable blue dye that is metabolized by themitochondria when added to the culture media. The dye then turns red intissue culture supernatants. The amounts of the red dye may be directlyquantitated by reading difference in optical densities between 570 nmand 600 nm. This reading reflects cellular activities and cell number.

[0478] Normal primary dermal keratinocytes (CC-0255, NHEK-Neo pooled)are purchased from Clonetics, Inc. These cells are passage 2.Keratinocytes are grown in complete keratinocyte growth media (CC-3001,KGM; Clonetics, Inc.) until they reach 80% confluency. The cells aretrypsinized according to the manufacturer's specification. Briefly,cells were washed twice with Hank's balanced salt solution. 2-3 ml oftrypsin was added to cells for abcut 3-5 min at room temperature.Trypsin neutralization solution was added and cells were collected.Cells are spun at 600×g for 5 min at room temperature and plated intonew flasks at 3,000 cells per square centimeter using pre-warmed media.

[0479] For the proliferation assay, plate 1,000-2,000 keratinocytes perwell of the Corning flat bottom 96-well plates in complete media exceptfor the outermost rows. Fill the outer wells with 200 μl of sterilewater. This helps to keep temperature and moisture fluctuations of thewells to the minimum. Grow cells overnight at 37° C. with 5% CO₂. Washcells twice with keratinocyte basal media (CC-3101, KBM, Clonetics,Inc.) and add 100 μl of KBM into each well. Incubate for 24 hours.Dilute growth factors in KBM in serial dilution and add 100 μl to eachwell. Use KGM as a positive control and KBM as a negative control. Sixwells are used for each concentration point. Incubate for two to threedays. At the end of incubation, wash cells once with KBM and add 100 μlof KBM with 10% v/v alamarBlue pre-mixed in the media. Incubate for 6 to16 hours until media color starts to turn red in the KGM positivecontrol. Measure O.D. 570 nm minus O.D. 600 nm by directly placingplates in the plate reader.

[0480] Results

[0481] Stimulation of Keratinocyte proliferation by KGF-2

[0482] To demonstrate that KGF-2 (i.e., starting at amino acid Cys37 asdescribed in Examples 7 and 8 above) and N-terminal deletion mutantsKGF-2Δ33 and KGF-2Δ28 were active in stimulating epidermal keratinocytegrowth, normal primary human epidermal keratinocytes were incubated withthe E. coli-expressed and purfied KGF-2 protein (batch number E3)(SEQ IDNO: 2), KGF-2Δ33 (batch number E1) and KGF-2Δ28 (batch number E2). TheKGF-2 protein stimulated the growth of epidermal keratinocytes with anEC50 of approximately 5 ng/ml, equivalent to that of FGF7/KGF-1 (FIG.21A). In contrast, other FGF's such as FGF-1 and FGF-2 did not stimulatethe growth of primary keratinocytes. The EC50 for KGF-2Δ33 was 0.2 ng/mland that for KGF-2Δ28 2 ng/ml (See FIGS. 21B and C). Thus, KGF-2appeared to be as potent as FGF7/KGF in stimulating the proliferation ofprimary epidermal keratinocytes. However, KGF-2Δ33 is more potent instimulating keratinocyte proliferation than the “Cys (37)” KGF-2described in Examples 7 and 8 above and the KGF-2Δ28.

[0483] Scarring of wound tissues involves hyperproliferation of dermalfibroblasts. To determine whether the stimulatory effects of KGF-2 wasspecific for keratinocytes but not for fibroblasts, mouse Balb.c.3T3fibroblsts and human lung fibroblasts were tested. Niether types offibroblasts responded to KGF-2 in proliferation assays. Therefore, KGF-2appeared to be a mitogen specific for epidermal keratinocytes but notmesenchymal cells such as fibroblasts. This suggested that thelikelyhood of KGF-2 causing scarring of the wound tissues was low.

EXAMPLE 11

[0484] A. Mitogenic Effects of KGF-2 on Cells Transfected with SpecificFGF Receptors

[0485] To determine which FGF receptor isoform(s) mediate theproliferative effects of KGF-2, the effects of KGF-2 on cells expressingspecific PGF receptor isoforms were tested according to the methoddescribed by Santos-Ocampo et al. J. Biol. Chem. 271:1726-1731 (1996).FGF7/KGF was known to induce mitogenesis of epithelial cells by bindingto and specifically activating the FGFR2iiib form (Miki et al. Science251:72-75 (1991)). Therefore, the proliferative effects of KGF-2 inmitogensis assays were tested using cells expressing one of thefollowing FGF receptor isoforms: FGFR1iiib, FGFR2iiib, FGFR3iiib, andFGFR4.

[0486] Mitogensis assay of cells expressing FGF receptors

[0487] Thymidine incorporation of BaF3 cells expressing specific FGFreceptors were performed as described by Santos-Ocampo et al. J. Biol.Chei. 271:1726-1731 (1996). Briefly, BaF3 cells expressing specific FGFreceptors were washed and resuspended in Dubeco's modified Eagle'smedium, 10% neonatal bovine serum, L-glutanime. Approximately 22,500cells were plated per well in a 96-well assay plate in media containing2 μg/ml Heparin. Test reagents were added to each well for a totalvolume of 200 μl per well. The cells were incubated for 2 days at 37° C.To each wll, 1 μCi of ³H-thymidine was then added in a volume of 50 μl.Cells were harvested after 4-5 hours by filteration through glass fiberpaper. Incorporated ³H-thyridine was counted on a Wallac beta platescintillaion counter.

[0488] Results

[0489] The results revealed that KGF-2 protein (Thr (36)-Ser (208) ofFIG. 1 (SEQ ID NO: 2) with a N-terminal Met added thereto) stronglystimulated the proliferation of Baf3 cells expressing the KGF receptor,FGFR2iiib isoform, as indicated by ³H-thymidine incorporation (FIG.22A). Interestingly, a slight stimulatory effect of KGF-2 on theproliferation of Baf3 cells expressing the FGFR1iiib isoform wasobserved. KGF-2 did not have any effects on cells expressing theFGFR3iiib or the FGFR4 forms of the receptor.

[0490] FGF7/KGF stimulated the proliferation of cells expressing the KGFreceptor, FGFR2iiib but not FGFR1iiib isoform. The difference betweenKGF-2 and FGF7/KGF was intriguing. In the control experiments, aFGFstimulated its receptors, FGFR1iiib and iiic and bFGF stimulated itsreceptor FGFR2iiic. Thus, these results suggested that KGF-2 binds toFGFR2iiib isoform and stimulates mitogenesis. In contrast to FGF7/KGF,KGF-2 also binds FGFR1iiib isoform and stimulates mitogenesis.

[0491] B. Mitogenic Effects of KGF-2Δ33 on Cells Transfected withSpecific FGF Receptors

[0492] As demonstrated above FGFs or KGF-1 and -2 both bind to andactivate the FGF 2iiib receptor (FGFR 2iiib). The proliferative effectsof KGF-2Δ33 in mitogensis assays were tested using cells expressing oneof the following FGF receptor isoforms: FGFR2iiib or FGFR2iiic (the2iiic receptor-transfected cells are included as a negative control).

[0493] The experiments were performed as above in part A of thisexample. Briefly, BaF3 cells were grown in RPMI containing 10% bovinecalf serum (BCS—not fetal serum), 10% conditioned medium from culturesof WEHI3 cells (grown in RPMI containing 5% BCS), 50 nMβ-mercaptoethanol, L-Glu (2% of a 100×stock) and pen/strep (1% of a100×stock).

[0494] For the assay, BaF3 cells were rinsed twice in RPMI mediumcontaining 10% BCS and 1 μg/ml heparin. BaF3 cells (22,000/well) wereplated in a 96-well plate in 150 μl of RPMI medium containing 10% BCSand 1 μg/ml heparin. Acidic FGF, basic FGF, KGF-1 (HG15400) or KGF-2proteins (HG03400, 03401, 03410 or 03411) were added at concentrationsfrom approximately 0 to 10 nM. The cells were incubated in a finalvolume of 200 μl for 48 hours at 37° C. All assays were done intriplicate. Tritiated thymidine (0.5 μCi) was added to each well for 4hours at 37° C. and the cells were then harvested by filtration througha glass fiber filter. The total amount of radioactivity incorporated wasthen determined by liquid scintillation counting. The following positivecontrols were used: basic FGF and acidic FGF for FGFR2iiic cells; acidicFGF and KGF-1 for FGFR2iiib cells. The following negative controls wereused: Basal medium (RPMI medium containing 10% BCS and 1 μg/ml heparin).

[0495] Results:

[0496] The results revealed that KGF-2 (Tbr (36)-Ser (208) withN-terminal Met added), KGF-2Δ33 and KGF-2Δ28 proteins stronglystimulated the proliferation of BaF3 cells expressing the KGF receptor,FGFR2iiib isoform, as indicated by ³H-thymidine incorporation (FIGS.22A-C). The KGF-2 proteins did not have any effects on cells expressingthe FGFR2iiic forms of the receptor. These results suggested that KGF-2proteins bind to FGFR2iiib isoform and stimulates mitogenesis. Inaddition, it appears that KGF-2Δ33 was able to stimulate theproliferation of the BaF3 cells better than the KGF-2 (Thr(36)-Ser(208)).

EXAMPLE 12

[0497] A. Construction of E. coli Optimized Full Length KGF-2

[0498] In order to increase expression levels of full length KGF-2 in anE. coli expression system, the codons of the amino terminal portion ofthe gene were optimized to highly used E. coli codons. For the synthesisof the optimized region of KGF-2, a series of six oligonucleotides weresynthesized: numbers 1-6 (sequences set forth below). These overlappingoligos were used in a PCR reaction for seven rounds at the followingconditions: Denaturation 95 degrees 20 seconds Annealing 58 degrees 20seconds Extension 72 degrees 60 seconds

[0499] A second PCR reaction was set up using 1 μl of the first PCRreaction with KFG-2 sythetic primer 6 as the 3′ primer and KGF-2synthetic 5′ BamHI as the 5′ primer using the same conditions asdescribed above for 25 cycles. The product produced by this finalreaction was restricted with AvaII and BamHI. The KGF-2 construct ofExample 1 was restricted with AvaII and HindIII and the fragment wasisolated. These two fragments were cloned into pQE-9 restricted withBamHI and HindIII in a three fragment ligation.

[0500] Primers used for constructing the optimized synthetic KGF-21/208: KGF-2 Synthetic Primer 1: KGF-2 Synthetic Primer 1:ATGTGGAAATGGATACTGACCCACTGCGCTTCTGCT (SEQ ID NO:31)TTCCCGCACCTGCCGGGTTGCTGCTGCTGCTGCTGC TGCTGCTGTTC KGF-2 Synthetic Primer2: CCGGAGAAACCATGTCCTGACCCAGAGCCTGGCAGG (SEQ ID NO:32)TAACCGGAACAGAAGAAACCAGGAACAGCAGCAGGA AGCAGCAGCA KGF-2 Synthetic Primer3: GGGTCAGGACATGGTTTCTCCGGAAGCTACCAACTC (SEQ ID NO:33)TTCTTCTTCTTCTTTCTCTTCTCCGTCTTCTGCTGG TCGTCACG KGF-2 Synthetic Primer 4:GGTGAAAGAGAACAGTTTACGCCAACGAACGTCACC (SEQ ID NO:34)CTGCAGGTGGTTGTAAGAACGAACGTGACGACCAGC AGAAGACGG KGF-2 Synthetic Primer 5:CGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTC (SEQ ID NO:35)CTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACC AAA KGF-2 Synthetic Primer 6:TTTGGTCCCAGAAACTTTACCGTTTTTTTCGATTTT (SEQ ID NO:36) CAG KGF-2 Synthetic5′ BamHI AAAGGATCCATGTGGAAATGGATACTGACCCACTGC (SEQ ID NO:37)

[0501] The resulting clone is shown in FIG. 23 (SEQ ID NOS: 38 and 39).

[0502] B. Construction of E. coli Optimized Mature KGF-2

[0503] In order to further increase expression levels of the mature formof KGF-2 in an E. coli expression system, the codons of the aminoterminal portion of the gene were optimized to highly used E. colicodons. To correspond with the mature form of KGF-1, a truncated form ofKGF-2 was constructed starting at threonine 36. E. coli synthetic KGF-2from Example 12 A was used as a template in a PCR reaction using BspHI5′ KGF-2 as the 5′ primer (sequence given below) and HindIII 3′ KGF-2 asthe 3′ primer (sequence given below). Amplification was performed usingstandard conditions as given above in Example 12 A for 25 cycles. Theresulting product was restricted with BspHI and HindII and cloned intothe E. coli expression vector pQE60 digested with NcoI and HindIII.BspHI 5′KGF-2 Primer: TTTCATGACTTGTCAAGCTCTGGGTCAAGATATGGT (SEQ IDNO:40) TC HindIII 3′ KGF-2 Primer: GCCCAAGCTTCCACAAACGTTGCCTTCC (SEQ IDNO:41)

[0504] The resulting clone is shown in FIG. 24A (SEQ ID NO: 42 and 43).

[0505] C. Construction of an Alternate E. coli Optimized Mature KGF 2

[0506] In order to further increase expression levels of the mature formof KGF-2 in an E. coli expression system, the codons of 53 amino acidsat the amino terminal portion of the E. coli optimized gene were changedto alternate highly used E. coli codons. For the synthesis of theoptimized region of KGF-2, a series of six oligonucleotides weresynthesized: numbers 18062, 18061, 18058, 18064, 18059, and 18063(sequences set forth below). These overlapping oligos were used in a PCRreaction for seven rounds at the following conditions: Denaturation 95degrees 20 seconds Annealing 58 degrees 20 seconds Extension 72 degrees60 seconds

[0507] Following the seven rounds of synthesis, a 5′ primer to thisregion, 18169 and a 3′ primer to this entire region, 18060, were addedto a PCR reaction, containing 1 microliter from the initial reaction ofthe six oligonucleotides. This product was amplified for 30 rounds usingthe following conditions: Denaturation 95 degrees 20 seconds Annealing55 degrees 20 seconds Extension 72 degrees 60 seconds

[0508] A second PCR reaction was set up to amplify the 3′ region of thegene using primers 18066 and 18065 under the same conditions asdescribed above for 25 rounds. The resulting products were separated onan agarose gel. Gel slices containing the product were diluted in 10 mMTris, 1mM EDTA, pH 7.5 One microliter each from each of diluted gelslices were used in an additional PCR reaction using primer 18169 as the5′ primer, and primer 18065 as the 3′ primer. The product was amplifiedfor 25 cycles using the same conditions as above. The product producedby this final reaction was and restricted with Eco R1 and HindIII, andcloned into pQE60, which was also cut with Eco R1 and HindIII (pQE6now). Sequences of the 5′ Synthetic Primers: 18169 KGF2 5′EcoRI/RBS:TCAGTGAATTCATTAAAGAGGAGAAATTAATCATGA [SEQ ID NO:44] CTTGCCAGG 18062 KGF2synth new R1 sense: TCATGACTTGCCAGGCACTGGGTCAAGACATGGTTT [SEQ ID NO:45]CCCCGCAAGCTA 18061 KGF2 synth R2 sense:GCTTCAGCAGCCCATCTAGCGCGCAGGTCGTCACGT [SEQ ID NO:46] TCGCTCTTACAACC 18058KGF2 Synth R3 sense: GTTCGTTGGCGCAAACTGTTCAGCTTTACCAAGTAC [SEQ ID NO:47]TTCCTGAAAATC 18066 KGF 2 20 bp Ava II sense:TCGAAAAAAACGGTAAAGTTTCTGGGAC [SEQ ID NO:48] 18064 KGF2 synth F1antisense: GATGGGCTGCTGAAGCTAGAGCTGGAGCTGTTGGTA [SEQ ID NO:49]GCTTCCGGGGAA 18059 KGF2 Synth F2 antisense:AACAGTTTGCGCCAACGAACATCACCCTGTAAGTGG [SEQ ID NO:50] TTGTAAGAG 18063 KGF2Synth F3 antisense: TTCTTGGTCCCAGAAACTTTACCGTTTTTTTCGATT [SEQ ID NO:51]TTCAGGAAGTA 18060 KGF 2 Ava II antisense: TTCTTGGTCCCAGAAACTTTACCG [SEQID NO:52] 18065 KGF2 HindIII 3′ Stop:AGATCAGGCTTCTATTATTATGAGTGTACCACCATT [SEQ ID NO:53] GGAAGAAAG

[0509] The sequence of the synthetic KGF-2 gene and it correspondingamino acid is shown in FIG. 24B (SEQ ID NO: 54 and 55)

EXAMPLE 13

[0510] Construction of KGF-2 Deletion Mutants

[0511] Deletion mutants were constructed from the 5′ terminus and 3′terminus of KGF-2 gene using the optimized KGF-2 construct from Example12 A as a template. The deletions were selected based on regions of thegene that might negatively affect expression in E. coli. For the 5′deletion the primers listed below were used as the 5′ primer. Theseprimers contain the indicated restriction site and an ATG to code forthe initiator methionine. The KGF-2 (FGF-12) 208 amino acid 3′ HindIIIprimer was used for the 3′ primer. PCR amplification for 25 rounds wasperformed using standard conditions as set forth in Example 12. Theproducts for the KGF-2 36aa/208aa deletion mutant were restricted BspHIfor the 5′ site and HindIII for the 3′ site and cloned into the pQE60which has bee digested with BspHI and HindIII. All other products wererestricted with NcoI for the 5′ restriction enzyme and HindIII for the3′ site, and cloned into the pQE60 which had been digested with NcoI andHindIII. For KGF.2 (FGF-12), 36aa/153aa and 128aa 3′ HindIII was used asthe 3′ primer with FGF-12 36aa/208aa as the 5′ primer. For FGF-1262aa/153aa, 128aa 3′ HindIII was used as the 3′ primer with FGF-1262aa/208aa as the 5′ primer. The nomenclature of the resulting clonesindicates the first and last amino acid of the polypeptide that resultsfrom the deletion. For example, KGF-2 36aa/153aa indicates that thefirst amino acid of the deletion mutant is amino acid 36 and the lastamino acid is amino acid 153 of KGF-2. Further, as indicated in FIGS.25-33, each mutant has N-terminal Met added thereto. Sequences of theDeletion Primers: FGF12 36aa/208aa: 5′BsphI GGACCCTCATGACCTGCCAGGCTCTGGG[SEQ ID NO:56] TCAGGAC FGF12 63aa/208aa: 5′NcoIGGACAGCCATGGCTGGTCGTCACGTTCG [SEQ D NO:57] FGF12 77aa/208aa: 5′NcoIGGACAGCCATGGTTCGTTGGCGTAAACTG [SEQ ID NO:58] FGF12 93aa/208aa: 5′NcoIGGACCCCCATGGAGAACTGCCCGTAGAGC [SEQ ID NO:59] FGF12 104aa/208aa: 5′NcoIGGACCCCCATGGAGAACTGCCCGTAGAGC [SEQ ID NO:60] FGF12 123aa/208aa: 5′NcoIGGACCCCCATGGTCAAAGCCATTAACAGC [SEQ ID NO:61] AAC FGF12 138aa/208aa:5′NcoI GGACCCCCATGGGGAAACTCTATGGCTCA [SEQ ID NO:62] AAAG FGF123′HindIII: (Used for all above deletion clones)CTGCCCAAGCTTATTATGAGTGTACCACCATTGGA [SEQ ID NO:63] AG FGF12 36aa/153aa:5′BspbI (as above) 3′HindIII CTGCCCAAGCTTATTACTTCAGCTTA [SEQ ID NO:64]CAGTCATTGT FGFI2 63aa/153aa: 5′NcoI and 3′HindIII, as above

[0512] The sequences for the resulting deletion mutations are set forthin FIGS. 25-33. [SEQ ID NOS: 65-82]

EXAMPLE 14

[0513] Construction of Cysteine Mutants of KGF-2

[0514] Construction of C-37 mutation primers 5457 5′ BsphI and 5258173aa 3′ HindIII were used to amplify the KGF-2 (FGF-12) template fromExample 12 A. Primer 5457 5′ BsphI changes cysteine 37 to a serine.Amplification was done using the standard conditions outlined above inExample 12 A for 25 cycles. The resulting product was restricted withBspHI and HindIII and cloned into E. coli expression vector pQE60,digested with BspHI and HindIII. (FIG. 34) [SEQ ID NO: 83]

[0515] For mutation of Cysteine 106 to serine, two PCR reactions wereset up for oligonucleotide site directed mutagenesis of this cysteine.In one reaction, 5453 BsphI was used as the 5′ primer, and 5455 was usedas the 3′ primer in the reaction. In a second reaction, 5456 was used asthe 5′ primer, and 5258 HindIII was used as the 3′ primer. The reactionswere amplified for 25 rounds under standard conditions as set forth inExample 12. One microliter from each of these PCR reactions was used astemplate in a subsequent reaction using, as a 5′ primer, 5453 BspHI, andas a 3′ primer, 5258 HindIII. Amplification for 25 rounds was performedusing standard conditions as set forth in Example 12. The resultingproduct was restricted with BspHI and HindIII and cloned into the E.coli expression vector pQE60, which was restricted with NcoI andHindIII.

[0516] Two PCR reactions were required to make the C-37/C-106 mutant.Primers 5457 Bsph1 and 5455 were used to create the 5′ region of themutant containing cysteine 37 to serine substitution, and primer 5456and 5258 HindIII were used to create the 3′ region of the mutantcontaining cysteine 106 to serine substitution. In the second reaction,the 5457 BsphI primer was used as the 5′ primer and the 5258 HindIIIprimer was used as the 3′ primer to create the C-37/C-106 mutant using 1μl from each of the initial reactions together as the template. This PCRproduct was restricted with BsphI and HindIII, and cloned into pQE60that had been restricted with NcoI and HindIII. The resulting clone isshown in FIG. 35 (SEQ ID NO: 84) Sequences of the Cysteine MutantPrimers: 5457 BspHI: GGACCCTCATGACCTCTCAGGCTCTGGGT (SEQ ID NO:85) 5456:AAGGAGAACTCTCCGTACAGC (SEQ ID NO:86) 5455; GCTGTACGGTCTGTTCTCCTT (SEQ IDNO:87) 5453 BspHI: GGACCCTCATGACCTGCCAGGCTCTGGGTCAGGAC (SEQ ID NO:88)5258 HindIII: CTGCCCAAGCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID NO:89)

EXAMPLE 15

[0517] Production and Purification of KGF-2 (FGF-12)

[0518] The DNA sequence encoding the optimized mature protein describedin Example 12 B (i.e., amino acids T36 through S208 of KGF-2) was clonedinto plasmid pQE-9 (Qiagen). E. coli (M15/rep4;Qiagen) were grown tostationary phase overnight at 37° C. in LB containing 100 μg/mlAmpicillin and 25 μg/ml Kanamycin. This culture was used to innoculatefresh LB media containing containing 100 μg/ml Ampicillin and 25 μg/mlKanamycin at a dilution of 1:50. The cells were grown at 37° C. to anO.D.₅₉₅ of 0.7, induced by the addition of isopropyl1-thio-b-D-galactopyranoside (IPTG) to a final concentration of 1 mM.After 3-4 hours, the cells were harvested by centrifugation, andresuspended in a buffer containing 60 mM NaPO₄ and 360 mM NaCl at aratio of 5 volumes of buffer: 1 volume of cell paste. After disruptionin a Mautin Gaulin, the extract was adjusted to pH to 8.0 by theaddition of NaOH and clarified by centrifugation. The clarified solubleextract was applied to a Poros HS-50 column (2.0×10.0 cm; PerSeptiveBiosystems, Inc.) and bound proteins step-eluted with 50 mM NaPO₄ pH 8.0containing 0.5 M, 1.0 M and 1.5 M NaCl. The KGF-2 eluted in the 1.5 Msalt fraction which was then diluted five-fold with 50 mM NaPO₄ pH 6.5to a final salt concentration of 300 mM. This KGF-2 containing fractionwas then passed sequentially over a Poros HQ-20 column (2.0×7.0 cm;PerSeptive Biosystems, Inc.) and then bound to a Poros CM-20 column(2.0×9.0 cm; PerSeptive Biosystems, Inc.). KGF-2 (FGF-12)-containingfractions that eluted at about 500 mM to about 750 mM NaCl were pooled,diluted and re-applied to an CM-20 column to concentrate. Finally, theprotein was seperated on a gel filtration column (S-75; Pharmacia) in 40mM NaOAC pH6.5; 150 mM NaCl (Batch E-5) Alternatively, the gelfiltration column was run in Phosphate Buffered Saline (PBS, Batch E-4).KGF-2 containing fractions were pooled and protein concentrationdetermined by Bio-Rad Protein Assay. Proteins were judged to be >90%pure by SDS-PAGE. Finally, endotoxin levels determined by LimulusAmebocyte Lysate Assay (Cape Cod Associates) were found to be ≦1 Eu/mg.Proteins prepared in this way were able to bind heparin which is ahallmark of FGF family members.

EXAMPLE 16

[0519] A. Construction of N-terminal deletion mutant KGF-2Δ33

[0520] To increase the level of expression of KGF2 in E. coli, and toenhance the solubilty and stability properties of E. coli expressedKGF2, a deletion variant KGF-2Δ33 (KGF-2 aa 69-208) (SEQ ID NO: 96)which removes the first 68 amino acids of the pre-processed KGF2 wasgenerated. The rationale for creating this deletion variant was based onthe following observations. Firstly, mature KGF2 (KGF-2 aa 36-208)contains an uneven number (three) of cysteine residues which can lead toaggregation due to intra-molecular disulphide bridge formation. The KGFΔ33 deletion variant contains only two cysteine residues, which reducesthe potential for intra-molecular disulphide bridge formation andsubsequent aggregation. A decrease in aggregation should lead to anincrease in the yield of active KGF2 protein. Secondly, the KGF Δ33deletion variant removes a poly-serine stretch which is not present inKGF-1 and does not appear to be important for activity, but may hinderexpression of the protein in E. coli. Thus, removal of the poly-serinestretch may increase expression levels of active KGF-2 protein. Thirdly,expression of KGF Δ33 in E. coli, results in natural cleavage of KGF-2between residues 68 and 69. Thus, it is anticipated that KGF2 Δ33 willbe processed efficiently and will be stable in E. coli.

[0521] Construction of KGF2Δ33 in pQE6

[0522] To permit Polymerase Chain Reaction directed amplification andsub-cloning of KGF2 Δ33 into the E. coli protein expression vector,pQE6, two oligonucleotide primers (5952 and 19138) complementary to thedesired region of KGF2 were synthesized with the following basesequence. Primer 5952: 5′ GCGGCACATGTCTTACAACCACCTGCAGGGTG (SEQ IDNO:91) 3′ Primer 19138: 5′ GGGCCCAAGCTTATGAGTGTACCACCAT 3′ (SEQ IDNO:92)

[0523] In the case of the N-terminal primer (5952), an AflIIIrestriction site was incorporated, while in the case of the C-terminalprimer (19138) a HindIII restriction site was incorporated. Primer 5952also contains an ATG sequence adjacent and in frame with the KGF2 codingregion to allow translation of the cloned fragment in E. coli, whileprimer 19138 contains two stop codons (preferentially utilized in E.coli) adjacent and in frame with the KGF2 coding region which ensurescorrect translational termination in E. coli.

[0524] The Polymerase Chain Reaction was performed using standardconditions well known to those skilled in the art and the nucleotidesequence for the mature KGF-2 (aa 36-208) (constructed in Example 12C)as template. The resulting amplicon was restriction digested with AflIIIand HindIII and subcloned into NcoI/HindIII digested pQE6 proteinexpression vector.

[0525] Construction of KGF2Δ33 in pHE1

[0526] To permit Polymerase Chain Reaction directed amplification andsubcloning of KGF2 Δ33 into the E. coli expression vector, pHE1, twooligonucleotide primers (6153 and 6150) corresponding to the desiredregion of KGF2 were synthesized with the following base sequence. Primer6153: 5′ CCGGCGGATCCCATATGTCTTACAACCACCTGC (SEQ ID NO:93) AGG 3′ Primer6150: 5′ CCGGCGGTACCTTATTATGAGTGTACCACCTTG (SEQ ID NO:94) G 3′

[0527] In the case of the N-terminal primer (6153), an NdeI restrictionsite was incorporated, while in the case of the C-terminal primer (6150)an Asp718 restriction site was incorporated. Primer 6153 also containsan ATG sequence adjacent and in frame with the KGF2 coding region toallow translation of the cloned fragment in E. coli, while primer 6150contains two stop codons (preferentially utilized in E. coli) adjacentand in frame with the KGF2 coding region which ensures correcttranslational termination in E. coli.

[0528] The Polymerase Chain Reaction was performed using standardconditions well known to those skilled in the art and the nucleotidesequence for the mature KGF-2 (aa 36-208) (constructed in Example 12C)as template. The resulting amplicon was restriction digested with NdeIand Asp718 and subcloned into NdeI/Asp718 digested pHE1 proteinexpression vector. Nucleotide sequence of KGF2 Δ33:ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGG (SEQ ID NO:95)CGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAA ATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAAT CGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAG AATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCAT AATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACACCTC TGCTCACTTTCTTCCAATGGTGGTACACTCATAAAmino Acid sequence of KGF Δ33: MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKK(SEQ ID NO:96) ENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQ MYVALNGKGAPRRGQKTRRKNTSAHFLPMVVHS

[0529] B. Construction of an Optimized KGF-2Δ33

[0530] In order to increase the expression levels of KGF2 Δ33 in E.coli, the codons of the complete gene were optimized to match those mosthighly used in E. coli. As the template utilised to generate the KGF2Δ33 was codon optimized within the N-terminal region, the C-terminalamino acids (84-208) required optimization.

[0531] Firstly, amino acids 172-208 were codon optimized to generateKGF2Δ33(s172-208). This was achieved by an overlapping PCR strategy.Oligonucleotides PM07 and PM08 (corresponding to amino acids 172-208)were combined and annealed together by heating them to 70° C. andallowing them to cool to 37° C. The annealed oligonucleotides were thenutilized as template for a standard PCR reaction which was directed byprimers PM09 and PM10. In a separate PCR reaction following standardconditions well known to those skilled in the art and using KGF2Δ33 astemplate, oligonucleotides PM05 (which overlaps with the Pst1 sitewithin coding region of KGF2) and PM11 were used to amplify the regionof KGF2 corresponding to amino acids 84-172. In a third PCR reaction,the product of the first PCR reaction (corresponding to codon optimizedamino acids 172-208) and the product of the second PCR reaction(corresponding to codon non-optimized amino acids 84-172) were combinedand used as template for a standard PCR reaction directed byoligonucleotides PM05 and PM10. The resulting amplicon was digested withPst1/HindIII and sub-cloned into Pst1/HindIII digested pQE6KGF2Δ33,effectively substituting the corresponding non codon optimized region,and creating pQE6KGF2Δ33(s172-208).

[0532] To complete the codon optimization of KGF2, a synthetic genecodon optimized for the region of KGF2 corresponding to amino acids84-172 was generated utilising overlapping oligonucleotides. Firstly,four oligonucleotides (PM31, PM32, PM33 and PM 34) were combined andseven cycles of the following PCR was performed: 94° C., 30 secs; 46.5°C., 30 secs; and 72° C., 30 secs.

[0533] A second PCR reaction directed by primers PM35 and PM36 was thenperformed following standard procedures, utilizing 1 μl of the first PCRreaction as template. The resulting codon optimized gene fragment wasthen digested with Pst1/Sal1 and subcloned into Pst1/Sal1 digestedpQE6KGF2Δ33(s172-208) to create a fully optimized KGF2 encoding gene,pQE6KGF2Δ33s.

[0534] To create an alternative E. coli protein expression vector,KGF2Δ33s was PCR amplified utilising primers PM102 and PM130 onpQE6KGF2Δ33s. The resulting amplicon was digested with NdeI and EcoRVand subcloned into the pHE1 expression vector which had been digestedwith NdeI and Asp718 (blunt ended) to create pHE1Δ33s.

[0535] Oligonucleotide Sequences used in construction of codon optimizedKGF2 Δ33s: KGF2 Δ33s: PM05: CAACCACCTGCAGGGTGACG (SEQ ID NO:97) PM07:AACGGTCGACAAATGTATGTGGCACTGAACGGTA (SEQ ID NO:98)AAGGTGCTCCACGTCGTGGTCAGAAAACCCGTCG TAAAAACACC PM08:GGGCCCAAGCTTAAGAGTGTACCACCATTGGCAG (SEQ ID NO:99)AAAGTGAGCAGAGGTGTTTTTACGACGGGTTTTC TGACCACG PM09:GCCACATACATTTGTCGACCGTT (SEQ ID NO:100) PM10: GGGCCCAAGCTTAAGAGTG (SEQID NO:101) PM11: GCCACATACATTTGTCGACCGTT (SEQ ID NO:102) PM31:CTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCT (SEQ ID NO:103)CCTTCACCAAATACTTCCTGAAAATCGAAAAAAC GGTAAAGTTTCTGGTACCAAG PM32:AGCTTTAACAGCAACAACACCGATTTCAACGGAG (SEQ ID NO:104)GTGATTTCCAGGATGGAGTACGGGCAGTTTTCTT TCTTGGTACCAGAAACTTTACC PM33:GGTGTTGTTGCTGTTAAAGCTATCAACTCCAACT (SEQ ID NO:105)ACTACCTGGCTATGAACAAGAAAGGTAAACTGTA CGGTTCCAAAGAATTTAACAAC PM34:GTCGACCGTTGTGCTGCCAGTTGAAGGAAGCGTA (SEQ ID NO:106)GGTGTTGTAACCGTTTTCTTCGATACGTTCTTTC AGTTTACAGTCGTTGTTAAATTCTTTGGAACCPM35: GCGGCGTCGACCGTTGTGCTGCCAG (SEQ ID NO:107) PM36:GCGGCCTGCAGGGTGACGTTCGTTGG (SEQ ID NO:108) PM102:CCGGCGGATCCCATATGTCTTACAACCACCTGCA (SEQ ID NO:109) GG PM130:CGCGCGATATCTTATTAAGAGTGTACCACCATTG (SEQ ID NO:110) Nucleotide sequenceof KGF2 Δ33(s172-208): ATGTCTTACAACCACCTGCAGGGTGACGTTCGTT (SEQ IDNO:111) GGCGTAAACTGTTCTCCTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGTACC AAGAAAGAAAACTGCCCGTACTCCATCCTGGAAATCACCTCCGTTGAAATCGGTGTTGTTGCTGTTAA AGCTATCAACTCCAACTACTACCTGGCTATGAACAAGAAAGGTAAACTGTACGGTTCCAAAGAATTTA ACAACGACTGTAAACTGAAAGAACGTATCGAAGAAAACGGTTACAACACCTACGCTTCCTTCAACTGG CAGCACAACGGTCGACAAATGTATGTGGCACTGAACGGTAAAGGTGCTCCACGTCGTGGTCAGAAAAC CCGTCGTAAAAACACCTCTGCTCACTTTCTCGCCAATGGTGGTACACTCTTAA Amino Acid Sequence of KGF2 Δ33(s172-208):MSYNHLQGDVRWRKLFSFTKYFLKJEKNGKVSGT (SEQ ID NO:112)KKENCPYSILEITSVEIGVVAVKAINSNYYLAMN KKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHGRQMYVALNGKGAPRGQKTRRKNTSAHFLPMV VHS

[0536] C. Construction of N-terminal deletion mutant KGF-24

[0537] To increase the level of expression of KGF2 in E. coli and toenhance the stability and solubility properties of E. coli expressedKGF2, a deletion variant KGF2Δ4 (amino acids 39-208) which removes thefirst 38 amino acids of pre-processed KGF2 was constructed, includingthe cysteine at position 37. As the resulting KGF2 deletion moleculecontains an even number of cysteines, problems due to aggregation causedby intra-molecular disulphide bridge formation should be reduced,resulting in an enhanced level of expresssion of active protein.

[0538] To permit Polymerase Chain Reaction directed amplification andsub-cloning of KGF2 Δ4 into the E. coli protein expression vector, pQE6,two oligonucleotide primers (PM61 and 19138) were synthesized with thefollowing base sequence. PM61: CGCGGCCATGGCTCTGGGTCAGGACATG (SEQ IDNO:113) 19138: GGGCCCAAGCTTATGAGTGTACCACCAT (SEQ ID NO:114)

[0539] In the case of the N-terminal primer (PM61), an NcoI restrictionsite was incorporated, while in the case of the C-terminal primer(19138) a HindIII restriction site was incorporated. PM61 also containsan ATG sequence adjacent and in frame with the KGF2 coding region toallow translation of the cloned fragment in E. coli, while 19138contains a stop codon (preferentially utilized in E. coli) adjacent toand in frame with the KGF2 coding region which ensures correcttranslational termination in E. coli.

[0540] The Polymerase Chain Reaction was performed using standardconditions well known to those skilled in the art and the full lengthKGF2 (aa 36-208) as template (constructed in Example 12C). The resultingamplicon was restriction digested with NcoI and HindIII and subclonedinto NcoI/HindIII digested pQE6 protein expression vector. NucleotideSequence of KGF2 Δ4: ATGGCTCTGGGTCAAGATATGGTTTCTCCGGAAG (SEQ ID NO:115)CTACCAACTCTTCCTCTTCCTCTTTCTCTTCCCC GTCTTCCGCTGGTCGTCACGTTCGTTCTTACAACCACCTGCAGGGT GACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGA AAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATA ACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCA ACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAA AGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCAT TTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGG AAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGG TGGTACACTCATAA Amino Acid Sequence ofKGF2Δ4: MALGQDMVSPEATNSSSSSFSSPSSAGRHVRSYN (SEQ ID NO:116)HLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKEN CPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNG RQMYVALNGKGAPRRGQKTRRKNTSAHFLPMVVH S

EXAMPLE 17

[0541] KGF-2Δ33 Stimulated Wound Healing in Normal Rat

[0542] To demonstrate that KGF-2Δ33 would accelerate the healingprocess, wound healing of excisional wounds were examined using thefollowing model.

[0543] A dorsal 6 mm excisional wound is created on Sprague Dawley rats(n=5) with a Keyes skin punch. The wounds are left open and treatedtopically with various concentrations of KGF-2 Δ33 (in 40 mM NaOAc and150 mM NaCl, pH 6.5 buffer) and buffer (40 mM NaOAc and 150 mM NaCl, pH6.5) for 4 days commencing on the day of wounding. Wounds are measureddaily using a calibrated Jameson caliper. Wound size is expressed insquare millimeters. On the final day wounds were measured and harvestedfor further analysis. Statistical analysis was done using an unpaired ttest (mean±SE). Evaluation parameters include percent wound closure,histological score (1-3 minimal cell accumulation, no granulation; 4-6immature granulation, inflammatory cells, capillaries; 7-9 granulationtissue, cells, fibroblasts, new epithelium 10-12 mature dermis withfibroblasts, collagen, epithelium), re-epithelialization andimmunohistochemistry.

[0544] At three days postwounding, treatment with KGF-2 Δ33 displayed adecrease in wound size (30.4 mm² at 4 μg, p=0.006, 33.6 mm² at 1 μg,p=0.0007) when compared to the buffer control of 38.9 mm². At day fourpostwounding, treatment with KGF-2 Δ33 displayed a decrease in woundsize (27.2 mm² at 0.1 μg p=0.02, 27.9 mm² at 0.4 μg p=0.04) whencompared to buffer control of 33.8 mm². At day five postwounding,treatment with KGF-2 Δ33 displayed a decrease in wound size (18.1) mm²at 4 μg p=0.02 when compared to buffer control of 25.1 mm². See FIG. 36.

[0545] Following wound harvest on day 5, additional parameters wereevaluated. KGF-2 Δ33 displayed an increase in the percentage of woundclosure at 4 μg (71.2%, p=0.02) when compared to buffer control 60.2%.Administration of KGF-2 Δ33 also results in an improvement inhistological score at 1 and 4 μg (8.4 at 1 μg p=0.005, 8.5 at 4 μgp=0.04) relative to buffer control of 6.4. Re-epithelialization was alsoimproved at 1 and 4 μg KGF-2 Δ33 (1389 μm at 1 μg p=0.007, 1220 μm at 4μg p=0.02) relative to the buffer control of 923 μm. See FIG. 37.

[0546] This study demonstrates that daily treatment with KGF-2 Δ33accelerates the rate of wound healing in normal animals as shown by adecrease in the gross wound area. In addition, the histologicalevaluation of wound samples and assessment of re-epithelialization alsoshow that KGF-2 Δ33 improves the rate of healing in this normal ratmodel.

EXAMPLE 18

[0547] KGF-2Δ33 Effect on Tensile Strength and Epidermal Thickness inNormal Rat

[0548] To demonstrate that KGF-2Δ33 would increase tensile strength andepidermal thickness of wounds the following experiment was performed.

[0549] A 2.5 cm full thickness midline incisional wound is created onthe back of male Sprague Dawley rats (n=8 or 9). Skin incision is closedusing 3 equidistant metal skin staples. Buffer (40 mM NaOAc and 150 mMNaCl, pH 6.5) or KGF-2 Δ33 (in 40 mM NaOAc and 150 mM NaCl, pH 6.5buffer) were topically applied at the time of wounding. Four woundstrips measuring 0.5 cm in width are excised at day 5. Specimens areused for the study of breaking strength using an Instron™ skintensiometer, hydroxyproline determination and histopathologicalassessment. Breaking strength was defined as the greatest force withheldby each wound prior to rupture. Statistical analysis was clone using anunpaired t test (mean±SE).

[0550] In an incisional skin rat model, topically applied KGF-2 Δ33exhibited a statistically significant increase in breaking strength,tensile strength and epidermal thickness as a result of a singleintraincisional application subsequent to wounding. In one study, thebreaking strength of KGF-2 treated wounds at 1, 4, and 10 μg wassignificantly higher when compared to the buffer controls (107.3 g at 1μg p=0.0006, 126.4 g at 4 μg p<0.0001, 123.8 g at 10, μg p<0.0001). SeeFIG. 38.

[0551] Epidermal thickness was assessed under light microscopy on MassonTrichrome sections. KGF-2 Δ33 treated wounds displayed increasedepidermal thickening (60.5μ at 1 μg, 66.51μ at 4 μg p=0.01, 59.6μ at 10μg) in contrast with the buffer control of 54.8μ. See FIG. 39.

[0552] These studies demonstrate that a single intraincisionalapplication of KGF-2 augments and accelerates the wound healing processcharacterized by an increase in breaking strength and epidermalthickness of incisional wounds.

EXAMPLE 19

[0553] KGF-2Δ33 Effect on Normal Rat Skin

[0554] In order to determine the effect of KGF-2 Δ33 on normal rat skinfollowing intradermal injection the following experiment was performed.

[0555] Male adult SD rats (n=3) received six intradermal injections ofeither placebo or KGF-2 Δ33 (in 40 mM NaOAc and 150 mM NaCl, pH 6.5buffer) in a concentration of 1 and 4 μg in 50 μl on day 0. Animals wereinjected with 5-2′-bromo-deoxyrudine (BrdU)(100 mg/kg i.p.) two hoursprior to sacrifice at 24 and 48 hours. Epidermal thickness was measuredfrom the granular layer to the bottom of the basal layer. Approximately,20 measurements were mode along the injection site and the meanthickness quantitated. Measurements were determined using a calibratedmicrometer on Masson Trichrome stained sections under light microscopy.BrdU scoring was done by two blinded observers under light microscopyusing the following scoring system: 0-3 none to minimal BrdU labeledcells; 4-6 moderate labeling; 7-10 intense labeled cells. Animals weresacrificed 24 and 48 hours post injection. Statistical analysis was doneusing an unpaired t test. (mean±SE).

[0556] KGF-2 Δ33 treated skin displayed increased epidermal thickeningat 24 hours (32.2μ at 1 μg p<0.001, 35.4μ at 4 μg p<0.0001) in contrastwith the buffer control of 27.1μ. At 48 hours KGF-2 Δ33 treated skindisplayed increased epidermal thickening (34.0μ at 1 μg p=0.0003, 42.4μat 4 μg p<0.0001) compared to buffer control of 27.8μ. See FIG. 40.KGF-2 Δ33 treated skin also displayed increased BrdU immunostaining at48 hours (4.73 at 1 μg p=0.07, 6.85 at 4 μg p<0.0001) compared to buffercontrol of 3.33. See FIG. 41.

[0557] These studies demonstrate that a intradermal injection of KGF-2augments and accelerates epidermal thickening. Thus, KGF-2 would haveapplications to prevent or alleviate wrinkles, improve aging skin andreduce scaring or improve healing from cosmetic surgery. In addition,KGF-2 can be used prophylactically to prevent or reduce oral mucosistis(mouth ulcers), intestinal inflammation in response to chemotherapy orother agents.

EXAMPLE 20

[0558] Anti-inflammatory Effect of KGF-2 on PAF-induced Paw Edema

[0559] To demonstrate an anti-inflammatory effect of KGF-2 the followingexperiment was performed using PAF-induced paw edema inflammation model.

[0560] Groups of four lewis rats (190˜210 gm) were injectedsubcutaneously in the foot pad of the right hind paw with 120 μlsolution containing 2.5 nMol of PAF, together with the followingreagents: 125 μg of Ckb-10(B5), 24 μg of LPS, 73 μg of KGF-2 (Thr(36)-Ser (208) of FIG. 1 (SEQ ID NO: 2) with a N-terminal Met) or noprotein. The left hind paws were given the same amount of buffer to useas parallel control. Paw volume was quantified immediately before, or 30and 90 minutes after PAF injection using a plethysmograph system.Percent (%) change of paw volume were calculated. Testing reagents inexperiment No. 1 and No. 2 Groups PAF(R.) Ckβ-10(R.) LPS(R.) KGF-2(R.)(N = 4) 2.5 nMol 1.04 mg/ml 200 μg/ml 0.73 mg/ml Buffer 1 20μl — — — 100μl 2 20 μl 100 μl — — — 3 20 μl — 100 μl — — 4 20 μl — — 100 μl —

[0561] As shown in FIG. 42, right hind paws injected with PAF aloneresulted in a significant increase in paw volume (75 or 100% forexperiment No. 1 or No. 2, respectively) at 0.5 hour post injection asexpected; while left hind paws receiving buffer or right hind pawsreceiving LPS or SEB alone show little sign of edema (data not shown).However, when KGF-2 was given together with PAF locally, there is asubstantial reduction (25 or 50% for experiment No. 1 or No. 2,respectively) in paw volume compared with PAF alone-challenged paws. Thereduction of paw edema was not observed in animal receiving PAF togetherwith Ckb-10 (a different protein), LPS or SEB (two inflammatorymediators). These results suggest that the anti-inflammatory effect ofKGF-2 is specific and not due to some non-specific nature of theprotein.

[0562] Effect of KGF-2 Δ33 on PAF-induced paw edema in rats

[0563] Following the experiments described above with KGF-2 Δ33 toconfirm its in vitro biological activities for stimulating keratinocyteproliferation and its in vivo effect on wound healing, KGF-2 Δ33 wasfurther evaluated in the PAF-induced paw edema model in rats. Groups offour Lewis rats (190-210 gm) were injected subcutaneously in the footpad of the right hind paw with 12 μl solution containing 2.5 nMol ofPAF, together with 210 μg of KGF-2 Δ33 or albumin. The left hind pawswere given the same amount of buffer, albumin or KGF-2 Δ33 alone to useas parallel control. Paw volume was quantified at different intervalsafter PAF injection using a plethysmograph system. Percent (%) change ofpaw volume was calculated.

[0564] As shown in FIG. 43, right hind paws injected with PAF andalbumin resulted in a significant increase (75%) in paw volume at 0.5hour post injection as expected; while left hind paws receiving buffer,albumin or KGF-2 Δ33 alone showed little sign of edema. However, whenKGF-2 Δ33 was given together with PAF locally, there was a substantialreduction (average 20%) in paw volume, when compared with PAF plusalbumin-challenged paws, throughout the entire experiment which wasended in 4 hours. These results confirm the anti-inflammatory propertyof KGF-2 Δ33. Testing Reagents Groups PAF Albumin KGF-2 Δ33 (N = 4) 2.5nMol 2.1 mg/ml 2.1 mg/ml Buffer 1 20 μl 100 μl — — 2 20 μl — 100 μl — 3— 120 μl — — 4 — — 120 μl — 5 — — — 120 μl

[0565] Thus, KGF-2 is useful for treating acute and chronic conditionsin which inflammation is a key pathogenesis of the diseases includingbut not limiting to psoriasis, eczema, dermatitis and/or arthritis.

EXAMPLE 21

[0566] Effect of KGF-2 Δ33 on End-to-End Colonic Anastomosis Rat Model

[0567] This example demonstrates that KGF-2 Δ33 will increase the rateof intestinal repair in a model of intestinal or colonic anastomosis inWistar or Sprague Dawley rats.

[0568] The use of the rat in experimental anastomosis is a wellcharacterized, relevant and reproducible model of surgical woundhealing. This model can also be extended to study the effects of chronicsteriod treatment or the effects of various chemotherapeutic regimens onthe quality and rate of surgical wound healing in the colon and smallintestine (Mastboom W. J. B. et al. Br, J. Surg. 78: 54-56 (1991), SalmR. et al. J Surg. Oncol. 47: 5-11, (1991), Weiber S. et al. Eur. Surg.Res. 26: 173-178 (1994)). Healing of anastomosis is similar to that ofwound healing elsewhere in the body. The early phases of healing arecharacterized by acute inflammation followed by fibroblast proliferationand synthesis of collagen. Collagen is gradually modeled and the woundis strengthened as new collagen is synthesized. (Koruda M. J., andRolandelli, R. H. J. Surg. Res. 48: 504-515 (1990). Most postoperativecomplications such as anastomotic leakage occur during the first fewdays following surgery—a period during which strength of the colon ismainly secured by the ability of the wound margin to hold sutures. Thesuture holding capacity of the GI tract has been reported to decrease byas much as 80% during the first postoperative days (Hogstrom H andHaglund U. Acta Chir Scand 151: 533-535 (1985), Jonsson K, et al. Am J.Surg. 145: 800-803 (1983)).

[0569] Male adult SD rats (n=5) were anesthetized with a combination ofketamine (50 mg/kg) and xylazine (5 mg/kg) intramuscularly. Theabdominal cavity was opened with a 4 cm long midline incision. A 1 cmwide segment of the left colon was resected 3 cm proximal to theperitoneal reflection while preserving the marginal vessels. A singlelayer end-to-end anatomosis was performed with 8-10 interrupted 5-0Vicryl inverted sutures to restore intestinal continuity. Theanastomosis was then topically treated via syringe with either buffer orKGF-2 Δ33 at concentrations of 1 and 4 μg. The incisional wound wasclosed with 3-0 running silk suture for the muscle layer and surgicalstaples for the skin. Treatments were then administered daily thereafterand consisted of buffer or KGF-2 Δ33 and 1 and 5 mg/kg sc. Weights weretaken on the day of surgery and daily thereafter. Animals wereeuthanized 24 hours following the last treatment (day 5). Animals wereanesthetized and received barium enemas and were x-rayed at a fixeddistance. Radiologic analysis following intracolonic administration by 2blinded observers revealed that KGF-2 Δ33 treated groups had 1) adecreased rate of barium leakage at the surgical site, 2) lesser degreeof constriction at the surgical site, and 3) an increase in the presenceof fecal pellets distal to the surgical site. Colonic AnastomosisRadiologic Analysis Feces Anastomotic Proximal Peritoneal Groups PresentConstriction Distension Leakage No Treatment  20% 80%  80% 60% (N = 5)Buffer  40% 60%  80% 75% (N = 5) KGF-2 Δ33 [1 mg/kg]  60% 20% 100% 20%(N = 5) KGF-2 Δ33 [5 mg/kg] 100%  0%  75% 25% (N = 4)

EXAMPLE 22

[0570] Construction of Carboxy Terminal Mutations in KGF-2

[0571] The carboxyl terminus of KGF-2 is highly charged. The density ofthese charged residues may affect the stability and consequently hesolubility of the protein. To produce muteins that might stabilize theprotein in solution a series of mutations were created in this region ofthe gene.

[0572] To create point mutants 194 R/E, 194 R/Q, 191 K/E, 191 K/Q,188R/E, 188R/Q, the 5952 KGFA33 5′ Afl III 5′ primer was used with theindicated 3′ primers, which contain the appropriate point mutations forKGFF-2, in PCR reactions using standard conditions well known to thoseskilled in the art with KGF-2Δ33 as template. The resulting productswere restricted with AflIII and Hind III and cloned into the E. coliexpression vector, pQE60 restricted with NcoI and Hind III. KGF2Δ33,194R/E Construction: The following primers were used: 5952KGFΔ33 5′ AflIII: 5′GCGGCACATGTCTTACAACCACCTGCAGGGTG 3′ (SEQ ID NO:117) KGF23′HindIII 194aa R to E: 5′CTGCCCAAGCTTTTATGAGTGTACCACCATTG (SEQ IDNO:118) GAAGAAAGTGAGCAGAGGTGTTTTTTTCTCGTGT TTTCTGTCC 3′ KGF2Δ33,194 R/ENucleotide sequence: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTT (SEQ ID NO:119)GGCGTAAACTGTTCTCTTTCACCAAATACTFCCT GAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAAC TGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGAGTTG TTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCA AAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAA ATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAGAAAAAAACA CCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG KGF2Δ33,194 R/E Amino acid sequence:MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGT (SEQ ID NO:120)KKENCPYSILEITSVEIGVVAVKAINSNYYLAMN KKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQKTR E KNTSAHFLP MVVHS KGF2 Δ33,194 R/QConstruction: The following primers were used: 5952 KGF Δ33 5′ Afl III:5′GCGGCACATGTCTTACAACCACCTGCAGGGTG 3′ (SEQ ID NO:121) KGF2 3′ HindIII194 aa R to Q: 5′CTGCCCAAGCTTTTATGAGTGTACCACCATTG (SEQ ID NO:122)GAAGAAAGTGAGCAGAGGTGTTTTTCTGTCGTGTTTT CTGTCC 3′ KGF2 Δ33,194 R/QNucleotide Sequence: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTT (SEQ ID NO:123)GGCGTAAACTGTTCTCTTTCACCAAATACTTCCT GAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGA TAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAAC AAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGA AAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGA ATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGACAGAAAAACACCTCTGCTCACTTTCTTCCA ATGGTGGTACACTCATAG KGF2 Δ33,194 R/QAmino Acid Sequence: MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGT (SEQ ID NO:124)KKENCPYSILEITSVEIGVVAVKAINSNYYLAMN KKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQKTR Q KNTSAHFLP MVVHS KGF2Δ33,191 K/E Construction:The following primers were used: 5952 KGF Δ33 5′ Afl III:5′GCGGCACATGTCTTACAACCACCTGCAGGGTG (SEQ ID NO:125) 3′ KGF2 3′HindIII191aa K to E 5′CTGCCCAAGCTTTTATGAGTGTACCACCATTG (SEQ ID NO:126)GAAGAAAGTGAGCAGAGGTGTTTTTCCTTTCGTG TTTCCTGTCCTCTCCTTGG 3′ KGF2Δ33,191K/E Nucleotide Sequence: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTT (SEQ IDNO:127) GGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACC AAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAA AGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTA ACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGG CAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGGAAAC ACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG KGF2Δ33,191 K/E Amino Acid Sequence:MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGT (SEQ ID NO:128)KKENCPYSILEITSVEIGVVAVKAINSNYYLAMN KKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQ E TRRKNTSAHFLP MVVHS KGF2 Δ33, 191 K/QConstruction: The following primers were used: 5952 KGFΔ33 5′ Afl III:5′GCGGCACATGTCTTACAACCACCTGCAGGGTG (SEQ ID NO:129) 3′ KGF2 3′ HindIII191aa K to Q 5′CTGCCCAAGCTTTTATGAGTGTACCACCATTG (SEQ ID NO:130)GAAGAAAGTGAGCAGAGGTGTTTTTCCTTCGTGT CTGCTGTCCTCTCCTTGG 3′ KGF2 Δ33, 191K/Q Nucleotide Sequence: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTT (SEQ IDNO:131) GGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACC AAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAA AGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTA ACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGG CAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGCAGAC ACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG KGF2 Δ33, 191 K/Q Amino Acid Sequence:MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGT (SEQ ID NO:132)KKENCPYSILELTSVEIGVVAVKAINSNYYLAMN KKGKLYGSKEFNNDCKLKFRIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQ Q TRRKNTSMWLPM VVHS KGF2Δ33, 188 R/E Construction:The following primers were used: 5952 KGFΔ33 5′ Afl III:5′GCGGCACATGTCTTACAACCACCTGCAGGGTG (SEQ ID NO:133) 3′ KGF2 3′ HindIII188aa R to E: 5′CTGCCCAAGCTTTTATGAGTGTACCACCATTG (SEQ ID NO:134)GAAGAAAGTGAGCAGAGGTGTTTTTCCTTCGTGT TTTCTGTCCTTCCCTTGGAGCTCCTTT 3′KGF2Δ33, 188R/E Nucleotide Sequence: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTT(SEQ ID NO:135) GGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACC AAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAA AGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTA ACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGG CAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGGAAGGACAGAAAAC ACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG KGF2Δ33, 188R/E Amino Acid Sequence:MYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTK (SEQ ID NO:136)KENCPYSILEITSVEIGVVAVKAINSNYYLAMNK KGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPR E GQKTRRKNTSAHFLPM VVHS KGF2Δ33, 188 R/Q Construction:The following primers were used: 5952 KGF Δ33 5′ Afl III:5′GCGGCACATGTCTTACAACCACCTGCAGGGTG (SEQ ID NO:137) 3′ KGF2 3′ HindIII188aa R to Q: 5′CTGCCCAAGCTTTTATGAGTGTACCACCATTG (SEQ ID NO:138)GAAGAAAGTGAGCAGAGGTGTTTTTCCTTCGTGT TTTCTGTCCCTGCCTTGGAGCTCCTTT 3′KGF2Δ33, 188 R/Q Nucleotide Sequence: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTT(SEQ ID NO:139) GGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACC AAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAA AGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAAGTCTATGGCTCAAAAGAATTTA ACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGG CAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGCAGGGACAGAAAAC ACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG KGF2Δ33, 188 R/Q Amino Acid Sequence:MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGT (SEQ ID NO:140)KKENCPYSILEITSVEIGVVAVKAINSNYYLAMN KKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPR Q GQKTRRKNTSAHFLP MVVHS

[0573] KGF2 Δ33, 183K/E Construction:

[0574] For mutation 183K/E, two PCR reactions were set up foroligonucleotide site directed mutagenesis of this lysine. In onereaction, 5952 KGFΔ33 5′ AflIII was used as the 5′ primer, and KGF2183aa K to E antisense was used as the 3′ primer in the reaction. In asecond reaction, KGF2 5′ 183aa K to E sense was used as the 5′ primer,and KGF2 3′ HindIII TAA stop was used as the 3′ primer. KGF-2 Δ33 wasused as template for these reactions. The reactions were amplified understandard conditions well known to those skilled in the art. Onemicroliter from each of these PCR reactions was used as template in asubsequent reaction using, as a 5′ primer, 5453 BsphI, and as a 3′primer, 5258 HindIII. Amplification was performed using standardconditions well known to those skilled in the art. The resulting productwas restricted with Afl III and HindIII and cloned into the E. coliexpression vector pQE60, which was restricted with NcoI and HindIII. Thefollowing primers were used: 5952 KGF Δ33 5′ Afl III:5′GCGGCACATGTCTTACAACCACCTGCAGGGTG (SEQ ID NO:141) 3′ KGF2 5′ 183aa K toE sense: 5′TTGAATGGAGAAGGAGCTCCA 3′ (SEQ ID NO:142) KGF2 183aa K to Eantisense: 5′TGGAGCTCCTTCTCCATTCAA 3′ (SEQ ID NO:143) KGF2 3′ HindIIITAA stop: 5′CTGCCCAAGCTT TTATGAGTGTACCACCATT (SEQ ID NO:144) GG 3′ KGF2Δ33, 183K/E Nucleotide Sequence: ATGTCTTACAACCACCTGCAGGGTGACGTTCGTT (SEQID NO:145) GGCGTAAACTGTTCTCTTTCACCAAATACTTCCT GAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAAC TGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTG TTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCA AAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAA ATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAG GCAAATGTATGTGGCATTGAATGGAGAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACA CCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG KGF2 Δ33, 183K/E Amino Acid Sequence:MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGT (SEQ ID NO:146)KKENCPYSILEITSVEIGVVAVKAINSNYYLAMN KKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNG E GAPRRGQKTRRKNTSAHFLP MVVHS

EXAMPLE 23

[0575] Effect of KGF-2 on Survival After Total Body Irradiation inBalb/c Mice

[0576] Ionizing radiation is commonly used to treat many malignancies,including lung and breast cancer, lymphomas and pelvic tumors (Ward, W.F. et al., CRC Handbook of Animal Models of pulmonary Disease, CRCPress, pp. 165-195 (1989)). However, radiation-induced injury (lung,intestine, etc.) limits the intensity and the success of radiationtherapy (Morgan, G. W. et al., Int. J Radiat. Oncol. Biol. Phys. 31:361(1995)). The gastrointestinal mucosa has a rapid cell cycle and isparticularly sensitive to cytotoxic agents (Potten, C. S., et al., In:Cytotoxic Insult to Tissue, Churchill Livingstone, pp. 105-152 (1983)).Some of the manifestations of intestinal radiation damage include acuteproctitis, intestinal fibrosis, stricture or fistula formation(Anseline, D. F. et al Ann. Surg. 194:716-724 (1981)). A treatment whichprotects normal structures from radiation without altering theradiosensisitivity of the tumor would be beneficial in the management ofthese disorders. Regardless of the irradiated area, the dose ofradiation is limited by the radiosensitivity of normal tissue.Complications following total or partial body irradiation includepneumonitis, fibrosis, gastro-intestinal injury and bone marrowdisorders.

[0577] Several cytokines including IL-1, TNF, IL-6, IL-12 havedemonstrated radioprotective effects following TBI (Neta, R. et al, J.Exp. Med. 173:1177 (1991)). IL-11 has been shown to protect smallintestinal mucosal cells after combined irradiation and chemotherapy(Du, X. X. et al., Blood 83:33 (1994)) and radiation-induced thoracicinjury (Redlich, C. A. et al. The Journal of Immunology 157:1705-1710(1996)).

[0578] Animals

[0579] All experiments were performed using BALB/c mice. Animals werepurchased at 6 weeks of age and were 7 weeks old at the beginning of thestudy. All manipulations were performed using aseptic techniques. Thisstudy was conducted according to the guidelines set forth by the HumanGenome Sciences, Inc., Institutional Animal Care and Use Committee whichreviewed and approved the experimental protocol.

[0580] KGF-2

[0581] The protein consists of a 141 amino acid human protein termedKGF-2 Δ33. This protein is a truncated isoform of KGF-2 that lacks thefirst 33 amino-terminal residues of the mature protein. The geneencoding this protein has been cloned into an E. coli expression vector.Fractions containing greater that 95% pure recombinant materials wereused for the experiment. KGF-2 was formulated in a vehicle containing 40mM Na Acetate+150 mM NaCl, pH 6.5. Dilutions were made from the stocksolution using the same vehicle.

[0582] Total Body Irradiation and Experimental Design

[0583] Mice were irradiated with 519 RADS (5.19 Gy) using a 68 Mark IShepherd Cesium Irradiator. The KGF-2 Δ33 was administered dailysubcutaneously, starting 2 days before irradiation and continuing for 7days after irradiation. Daily weights were obtained in all mice. Groupsof mice were randomized to receive one of three treatments: Total bodyirradiation (TBI) plus buffer, TBI plus KGF-2 Δ33 (1 mg/kg sq), TBI plusKGF-2 Δ33 (5 mg/kg sq). Two independent experiments were performed.

[0584] Results

[0585] Two studies were performed using irradiated animals. In the firststudy, animals were irradiated with 519 RADS (5.19 Gy). Animals weretreated with buffer or KGF-2 Δ33 at 1 & 5 mg/kg, s.q. two days prior toirradiation and daily thereafter for 7 days. At day 25 after total bodyirradiation 1/5 animals survived in the buffer group. In contrast, KGF-2treated groups had 5/5 animals @ 1 mg/kg and 4/5 @ 5 mg/kg (FIG. 44).

[0586] In addition, KGF-2 treated animals displayed 0.9% and 5.3%,weight gain at day 20 post-TBI. In contrast, the buffer treated grouphad 4.2% weight loss at day 20. Normal non-irradiated age matchedcontrol animals showed 6.7% weight gain in the same time period (FIG.45).

[0587] Animals in the second study were also irradiated with 519 RADS(5.19 Gy). These animals were treated with buffer or KGF-2 Δ33 at 1 & 5mg/kg, s.q. two days prior to irradiation and daily thereafter for 7days. At day 15 after total body irradiation all the animals in thebuffer group were dead. KGF-2 at 1 mg/kg had 30% survival and 60%survival at 5 mg/kg. At day 25 after TBI the 1 mg/kg group showed 20%survival and the 5 mg/kg 50% survival (FIG. 46).

[0588] Conclusions

[0589] In summary, these results demonstrate that KGF-2 has protectiveeffect after TBI. The ability of KGF-2 to increase survival rate ofanimals subjected to TBI suggests that it would also be useful inradiation-induced injuries and to increase the therapeutic ratio ofirradiation in the treatment of malignancies.

EXAMPLE 24

[0590] Evaluation of KGF-2 in the TPA Model of Cutaneous Inflammation inMice

[0591] To demonstrate that KGF-2 would attenuate the progression ofcontact dermatitis, a tetradecanoylphorbol acetate (TPA)-inducedcutaneous inflammation model in mice is used. The use of the femaleBALB/c and male Swiss Webster mice in experimental cutaneousinflammation are well-characterized, relevant and reproducible models ofcontact dermatitis. These strains of mice have been shown to develop along-lasting inflammatory response, following topical application ofTPA, which is comprised of local hemodynamics, vascular permeability andlocal migration of leukocytes, and these pathological changes aresimilar to those of human dermatitis (Rao et al. 1993, Inflammation17(6):723; Rao et al. 1994, J. Kipid Mediators Cell Signalling 10:213).

[0592] Groups of mice receive either vehicle or KGF-2 intraperitoneally,sub-cutaneously, or intravenously 60 min after the topical applicationof TPA (4 μg/ear) applied as a solution in acetone (200 μg/ml), 10 μleach to the inner and outer surface of ear. The control group receives20 μl of acetone as a topical application. Four hours following theapplication of TPA, increase in ear thickness is measured and ears areexcised for histology. To determine vascular permeability in response toTPA, mice are intravenously injected through tail veins with Evans blue(300 mg/kg) at selected times after topical application of TPA and miceare sacrificed 15 min thereafter. Ears are excised and removed, thenextracted into dimethylformamide and centrifuged. Absorbance readingsare spectrophotometrically measured at 590 nm.

EXAMPLE 25

[0593] Effect of KGF-2 Δ33 in Wound Healing

[0594] The biological effects of KGF-2 Δ33 in the skin were examinedbased on the initial in vitro data demonstrating KGF-2′ s capacity tostimulate primary human epidermal keratinocytes as well as murine pro-BBaF3 cells transfected with the FGFR isoform 2iiib. Initial experimentswere performed to determine the biological effects of KGF-2 Δ33following intradermal administration. Following the intradermal studies,KGF-2 Δ33 was explored in a variety of wound healing models (includingfull thickness punch biopsy wounds and incisional wounds) to determineits potential as a wound healing agent.

[0595] Effect of KGF-2 Δ33 in a Glucocorticoid-Impaired Rat Model ofWound Healing

[0596] Impaired wound healing is an important clinical problemassociated with a variety of pathologic conditions such as diabetes andis a complication of the systemic administration of steroids orantimetabolites. Treatment with systemic glucocorticoids is known toimpair wound healing in humans and in animal models of tissue repair. Adecrease in circulating monocyte levels and an inhibition of procollagensynthesis have been observed subsequent to glucocorticoidadministration. The inflammatory phase of healing and matrix synthesisare therefore important factors involved in the complex process oftissue repair. In the present study the effects of multiple topicalapplications of KGF-2 were assessed on full thickness excisional skinwounds in rats in which healing has been impaired by the systemicadministration of methylprednisolone.

[0597] Sprague Dawley rats (n=5/treatment group) received 8 mm dorsalwounds and methylprednisolone (17 mg/kg, i.m.) to impair healing. Woundswere treated topically each day with buffer or KGF-2 at doses of 0.1,0.5 and 1.5 μg in a volume of 50 μl. Wounds were measured on days 2,4,6,and 8 using a calibrated Jameson caliper. On day 6 (data not shown), andday 8 (FIG. 47) KGF-2 treated groups showed a statistically significantreduction in wound closure when compared to the buffer control.

[0598] Effect of KGF-2 Δ33 on Wound Healing in a Diabetic Mouse Model

[0599] Genetically diabetic homozygous female (db+/db+) mice, 6 weeks ofage (n=6), weighing 30-35 g were given a dorsal full thickness woundwith a 6 mm biopsy punch. The wounds were left open and treated dailywith placebo or KGF-2 at 0.1, 0.5 and 1.5 μg. Wound closure wasdetermined using a Jameson caliper. Animals were euthanized at day 10and the wounds were harvested for histology.

[0600] KGF-2 displayed a significantly improvement in percent woundclosure at 0.1 μg (p0.02) when compared to placebo or with the untreatedgroup. Administration of KGF-2 also resulted in an improvement inhistological score at 0.1 μg (p=0.03) when compared to placebo or withthe untreated group (p=0.01) and 1.5 μg (p=0.05) compared to theuntreated group.

[0601] Conclusions

[0602] Based on the results presented above, KGF-2 shows significantactivity in impaired conditions such as glucocorticoid administrationand diabetes. Therefore, KGF-2 may be clinically useful in stimulatinghealing of wounds after surgery, chronic ulcers in patients withdiabetes or poor circulation (e.g., venous insufficiency and venousulcers), burns and other abnormal wound healing conditions such asuremia, malnutrition, vitamin deficiencies and systemic treatment withsteroids and antineoplastic drugs.

EXAMPLE 26

[0603] Effects of KGF-2 Δ33 on Oral Mucosa

[0604] Cytotoxic agents used clinically have the unfortunate effect ofinhibiting the proliferation of the normal epithelia in some locations,such as the oral mucosa, leading to life-threatening disturbances in themucosal barrier. We have conducted studies to examine the efficacy ofKGF-2 in this clinical area. The is data supports a therapeutic effectof KGF-2 in models of mucositis.

[0605] Effects of KGF-2 Δ33 on Hamster Oral Mucosa

[0606] We sought to determine if KGF-2 might induce proliferation ofnormal oral mucosal epithelium. The effect of KGF-2 in the oral mucosawas assessed in male Golden Syrian hamsters. The cheek pouch of thehamster was treated daily with buffer or KGF-2 Δ3 3 (at 0. 1, 1 and 10μg/cheek) which were applied topically to anesthetized hamster cheeks ina volume of 100 μl per cheek. The compound was in contact with the cheekfor a minimum of 60 seconds and subsequently swallowed. After 7 days oftreatment, animals were injected with BrdU and sacrificed as describedabove. Proliferating cells were labeled using anti-BrdU antibody. FIG.48 shows that there was a significant increase in BrdU labeling (cellproliferation) when animals were treated with 1 μg and 10 μg of KGF-2Δ33 (when compared to buffer treatment).

[0607] Topical treatment with KGF-2 induced the proliferationi of normalmucosal epithelial cells. Based upon these results, KGF-2 may beclinically useful in the prevention of oral mucositis caused by anychemotherapeutic agents (or other toxic drug regimens), radiationtherapy, or any combined chemotherapeutic-radiation therapy regimen. Inaddition, KGF-2 may be useful as a therapeutic agent by decreasing theseverity of damage to the oral mucosa as a result of toxic agents(chemotherapy) or radiotherapy.

EXAMPLE 27

[0608] The Effect of KGF-2 Δ33 on Ischemic Wound Healing in Rats

[0609] The aim of the experiments presented in this example was todetermine the efficacy of KGF-2 in wound healing using an ischemic woundhealing model.

[0610] The blood supply of local skin was partially interrupted byraising of a single pedicle full-thickness random myocutaneous flap (3×4cm). A full-thickness wound was made into the local skin, which iscomposed of the myocutaneous flap. Sixty, adult Sprague-Dawley rats wereused and randomly divided into treatments of KGF-2 Δ33 and placebogroups for this study (5 animals/group/time-point). The wounds wereharvested respectively at day 1, 3, 5, 7, 1 0 and 15 post-wounding.

[0611] The wound breaking strength did not show a significant differencebetween KGF-2 and buffer treated groups at early time points until day10 and 15 post-wounding.

[0612] The results indicated that KGF-2 improved significantly the woundbreaking strength in ischemic wound repair after 10 days post-wounding.These results also suggest that ischemia delays the healing process inboth groups compared to the data previously obtained in studies ofnormal wound healing.

[0613] This myocutaneous flap model supplies data and information in anischemic situation which results from venous return. These resultssuggest that KGF-2 could be used in the treatment of chronic venous legulcers caused by an impairment of venous return and/or insufficiency.

EXAMPLE 28

[0614] Evaluation of KGF-2 in the Healing of Colonic Anastomosis in Rats

[0615] The results of the present experiment demonstrate that KGF-2 Δ33increases the rate of intestinal repair in a model of intestinal orcolonic anastomosis in Wistar or Sprague Dawley rats. In addition, thismodel can be used to demonstrate that KGF-2 and its isoforms increasethe capability of the gastrointestinal or colon wall to bind sutures.

[0616] The use of the rat in experimental anastomosis is a wellcharacterized, relevant and reproducible model of surgical woundhealing. This model can also be extended to study the effects of chronicsteroid treatment or the effects of various chemotherapeutic regimens onthe quality and rate of surgical wound healing in the colon and smallintestine (Mastboom, W. J. B. et al. ,Br. J. Surg. 78:54-56 (1991);Salm, R. et al., J. Surg. Oncol 47:5-11 (1991); Weiber, S. et al., Eur.Surg. Res. 26:173-178 (1994)). Healing of anastomosis is similar to thatof wound healing elsewhere in the body. The early phases of healing arecharacterized by acute inflammation followed by fibroblast proliferationand synthesis of collagen. Collagen is gradually modeled and the woundis strengthened as new collagen is synthesized (Koruda, M. J., andRolandelli, R. H., J. Surg. Res. 48:504-515 (1990)). Most postoperativecomplications such as anastomotic leakage occur during the first fewdays following surgery—a period during which strength of the colon ismainly secured by the ability of the wound margin to hold sutures. Thesuture holding capacity of the GI tract has been reported to decrease byas much as 80% during the first postoperative days (Hogstrom, H. andHaglund, U., Acta Chir. Scand. 151:533-535 (1985); Jonsson, K. et al.,Am J Surg. 145:800-803 (1983)).

[0617] Rats were anesthetized with a combination of ketamine (50 mg/kg)and xylazine (5 mg/kg) intramuscularly. Animals were kept on a heatingpad during skin disinfection, surgery, and post-surgery. The abdominalcavity was opened with a 4 cm long midline incision. A 1 cm wide segmentof the left colon was resected 3 cm proximal to the peritonealreflection while preserving the marginal blood vessels. A single layerend-to-end anastomosis was performed with 8-10 interrupted 8-0 propyleneinverted sutures which were used to restore intestinal continuity. Theincisional wound was closed with 3-O running silk suture for the musclelayer and surgical staples for the skin. Daily clinical evaluations wereconducted on each animal consisting of individual body weight, bodytemperature, and food consumption patterns.

[0618] KGF-2 Δ33 and placebo treatment were daily administered sc,topically, ip, im, intragastrically, or intracolonically immediatelyfollowing surgery and were continued thereafter until the day ofsacrifice, day 7. There was an untreated control, a placebo group, andKGF-2 Δ33 groups. Two hours prior to euthanasia, animals were injectedwith 100 mg/kg BrdU i.p. Animals were euthanized 24 hours following thelast treatment (day 5). A midline incision was made on the anteriorabdominal wall and a 1 cm long colon segment, including the anastomosis,was removed. A third segment at the surgical site was taken for totalcollagen analysis.

[0619] In a series of two experiments, male adult SD rats (n=5) wereanaesthetized and received a single layer end-to-end anastomosis of thedistal colon with 8-10 interrupted 6-0 prolene inverted sutures. Theantstomotic site was then topically treated via syringe with eitherbuffer or KGF-2 Δ33 at concentrations of 1 and 4 μg. Animals were thentreated daily thereafter with either buffer or KGF-2 Δ33 atconcentrations of 1 mg/kg or 5 mg/kg ip. Animals were euthanized on day5 and the colon excised and snap frozen in liquid nitrogen, lyophilizedand subjected to collagen determinations. Collagen concentration isexpressed as μg collagen/mg dry weight tissue. Statistical analysis wasdone using an unpaired t test. Mean±SE. On day 5 rats were anesthetizedand subjected to barium enemas followed by radiographic analysis. Bariumenema radiologic assessment of end-to-end left colonic anastomosis fromtwo experiments showed a consistent reduction in peritoneal leakage withKGF-2 treated animals at 1 and 5 mg/kg. This data is shown in the Tablebelow. In addition, breaking strength at the site of surgery was alsoexamined using a tensiometer. No significant differences were observedbetween the KGF-2 Δ33 and buffer groups. As shown in FIG. 49,significant increases in collagen content at the surgical site weredemonstrated at both 1 mg/kg KGF-2 Δ33 (p=0.02) and 5 mg/kg (p=0.004)relative to buffer controls. TABLE Colonic Anastomosis RadiologicAnalysis Feces Anastomotic Peritoneal Groups Present Constriction*Leakage No Treatment 50% 2.0 75% (N = 8) Buffer 57% 1.0 50% (N = 7)KGF-2Δ33 [1 mg/kg] 50% 1.3 37% (N = 8) KGF-2Δ33 [5 mg/kg] 77% 1.6 11% (N= 9) # with a combination of ketamine (50 mg/kg) and xylazine (5 mg/kg)intramuscularly. The abdominal cavity was opened with a 4 cm longmidline incision. # A 1 cm wide segment of the left colon was resected 3cm proximal to the peritoneal reflection while preserving the marginalvessels. A single layer # end-to-end anastomosis was performed with 8-10interrupted 6-0 prolene inverted sutures to restore intestinal,continuity. The anastomosis was # then topically treated via syringewith either buffer or KGF-2 at concentrations of 1 and 4 μg. Theincisional wound was closed with 3-O running # silk suture for themuscle layer and surgical staples for the skin. Treatments were thenadministered daily thereafter and consisted of buffer or # KGF-2 Δ33 at1 and 5 mg/kg sc. Weights were taken on the day of surgery and dailythereafter. Animals were euthanized 24 hours following the # lasttreatment (day 5). Animals were anesthetized and received barium enemasand were x-rayed at a fixed distance. The anastomosis was then excised #for histopathological and biomechanical analysis.

EXAMPLE 29

[0620] Evaluation of KGF-2 in a Model of Inflammatory Bowel Disease

[0621] KGF-2 is a protein that induces keratinocyte proliferation invitro and is active in a variety of wound healing models in vivo. Thepurpose of this study was to determine whether KGF-2 was efficacious ina model of murine colitis induced by ad libitum exposure to dextransodium sulfate in the drinking water.

[0622] Six to eight week old female Swiss Webster mice (20-25 g, CharlesRiver, Raleigh, N.C.)) were used in a model of inflammatory boweldisease induced with a 4% solution of sodium sulfate (DSS, 36,000-44,000MW , American International Chemistry, Natick, Mass.)) administered adlibitum for one week. KGF-2 was given by daily parenteral administration(n=10). Three parameters were used to determine efficacy: 1) clinicalscore, based on evaluation of the stool; 2) histological score, based onevaluation of the colon; and 3) weight change. The clinical score wascomprised of two parts totaling a maximum of score of four. Stoolconsistency was graded as: 0=firm; 1=loose; 2 diarrhea. Blood in thestool was also evaluated on a 0 to 2 scale with 0=no blood; 1=occultblood; and 2=gross rectal bleeding. A mean group score above 3 indicatedprobable lethality, and disease which had progressed beyond itstreatable stage. Clinical scores were taken on Day 0, 4, 5, 6, and 7. Toarrive at a histological score, slides of the ascending, transverse anddescending colon were evaluated in a blinded fashion based oninflammation score (0-3) and crypt score (0-4). Body weight was measureddaily. Data was expressed as mean±SEM. An unpaired Student's t test wasused to determine significant differences compared to the diseasecontrol (* p<0.05; ** p<0.01; *** p<0.001).

[0623] When DSS-treated mice were given a daily, intra-peritoneal (IP)injection of KGF-2 Δ33 at a dose of 1, 5 or 10 mg/kg for 7 days, KGF-2significantly reduced clinical score, 28, 38 and 50 percent,respectively. Histological evaluation closely paralleled the dosedependent inhibition of the clinical score, with the 1, 5 and 10 mg/kgdose reducing histological score a significant 26, 48 and 51 percent.KGF-2 also significantly reduced weight loss associated with DSS-inducedcolitis.

[0624] In a second study, a comparison was made of the relative efficacyof KGF-2 Δ33 (10 mg/kg) when given IP or sub-cutaneous (SC) daily. Bythe end of the experiment on Day 7, animals injected IP with KGF-2 had asignificant, 34 percent reduction in clinical score while KGF-2 injectedSC resulted in a significant 46 percent reduction. The SC dose alsosignificantly reduced weight loss over DSS controls. Based onmeasurement of clinical score and body weight, SC administration ofKGF-2 is at least as efficacious as IP administration.

EXAMPLE 30

[0625] Effects of KGF-2 Δ33 on Normal Urinary Bladder and Prostate andin Cyclophosphamide-Induced Hemorrhagic Cystitis in Rats

[0626] The purpose of this example is to show that KGF-2 Δ33 is capableof stimulating urinary bladder proliferation in normal rats and thatthere is a therapeutic effect of KGF-2 Δ33 in a rat model ofCyclophosphamide-induced hemorrhagic cystitis.

[0627] Some cytotoxic agents used clinically have side effects resultingin the v inhibition of the proliferation of the normal epithelium in thebladder, leading to potentially life-threatening ulceration andbreakdown in the epithelial lining of the bladder. For example,cyclophosphamide causes hemorrhagic cystitis in some patients, acomplication which can be severe and in some cases fatal. Fibrosis ofthe urinary bladder may also develop with or without cystitis. Thisinjury is thought to be caused by cyclophosphamide metabolites excretedin the urine. Hematuria caused by cyclophosphamide usually is presentfor several days, but may persist. In severe cases medical or surgicaltreatment is required. Instances of severe hemorrhagic cystitis resultin discontinued cyclophosphamide therapy. In addition, urinary bladdermalignancies generally occur within two years of cyclophosphamidetreatment and occurs in patients who previously had hemorrhagic cystitis(CYTOXAN (cyclophosphamide) package insert). Cyclophosphamide has toxiceffects on the prostate and male reproductive systems. Cyclophosphamidetreatment can result in the development of sterility, and result in somedegree of testicular atrophy.

[0628] Effects of KGF-2 d33 on Normal Bladder, Testes and ProstateExperimental Design

[0629] Male Sprague-Dawley rats (160-220 g), (n=4 to 6/treatment group)were used in these studies. KGF-2 Δ33 was administered at a dose of 5mg/kg/day. Daily ip or sc injections of recombinant KGF-2 Δ33 or buffer(40 mM sodium acetate+150 mM NaCl at pH 6.5) were administered for aperiod of 1-7 days and the rats were sacrificed the following day. Toexamine the reversibility of effects induced with KGF-2 Δ33, additionalanimals were injected ip daily for 7 days with KGF-2 Δ33 or buffer andsacrificed after a 7 day treatment-free period.

[0630] On the day of sacrifice, rats were injected ip with 100 mg/kg ofBrdU. Two hours later the rats were overdosed with ether and selectedorgans removed. Samples of tissues were fixed in 10% neutral bufferedformalin for 24 hours and paraffin embedded. To detect BrdUincorporation into replicating cells, five micron sections weresubjected to immunohistochemical procedures using a mouse anti-BrdUmonoclonal antibody and the ABC Elite detection system. The sectionswere lightly counterstained with hematoxylin.

[0631] Sections were read by blinded observers. The number ofproliferating cells was counted in 10 random fields per animal at a 1×magnification for the prostate. To assess the effects of KGF-2 Δ33 inthe bladder, cross-sections of these tissues were prepared and thenumber of proliferating and non-proliferating cells were counted in tenrandom fields at 20× magnification. The results are expressed as thepercentage of labeled to unlabeled cells. Data are presented asmean+SEM. Statistical analyses (two-tailed unpaired t-test) wereperformed with the StatView Software Package and statisticalsignificance is defined as p <0.05.

[0632] Results

[0633] Bladder.

[0634] Intraperitoneal injection of KGF-2 Δ33 induced proliferation ofbladder epithelial cells over the 7 day study period (solid squares,FIG. 52) but this did not influence the weight of the organ.Subcutaneous administration elicited a small increase in proliferationbut this failed to achieve statistical significance (solid circles, FIG.52).

[0635] Prostate and Testes.

[0636] Both sc and ip administration of KGF-2 Δ33 induced significantproliferation of the prostate (FIG. 53) but this normalized after twoinjections. Prolonged ip treatment with KGF-2 Δ33 did not increase theweight of the prostate or testes.

[0637] Effects of KGF-2 Δ33 on Cyclophosphamide-Induced HemorrhagicCystitis

[0638] Experimental Design

[0639] Male Sprague Dawley rats (300-400 g) (n=5/group) were injectedi.v. via the tail vein with buffer placebo or KGF-2 Δ33 atconcentrations of 1 or 5 mg/kg 24 hours prior to a 200 mg/kg i.p.injection of cyclophosphamide. On the final day, 48 hours aftercyclophosphamide injection, rats were injected ip with 100 mg/kg ofBrdU. Two hours later the rats were killed by CO₂ administration.Fixation of the bladder was done by direct injection of 10% formalininto the lumen of the bladder and rinsing of the exterior of the bladderwith formalin. After 5 minutes, the bladder and prostate were removed.The urinary bladder and prostate gland were paraffin embedded,cross-sectioned and stained with H&E and a mouse anti-BrdU monoclonalantibody. The extent of urothelial damage was assessed using thefollowing scoring system: Bladders were graded by two independentobservers to describe the extent of the loss of urothelium. (Urothelialdamage was scored as 0, 25%, 50%, 75% and 100% loss of the urothelium).In addition, the thickness of the bladder wall was measured at 10 randomsites per section and expressed in gm.

[0640] Results

[0641] Macroscopic Observations

[0642] In rats treated with placebo and cyclophosphamide, bladders werethick and rigid. Upon injection of 10% formalin, very little expansionof the bladders was noted. However, in the groups pretreated with KGF-2Δ33, a greater elasticity of the bladder was noted upon direct injectionwith formalin suggesting a lesser degree of fibrosis.

[0643] Microscopic Observations

[0644]FIG. 54 shows the results of KGF-2 Δ33 pretreatment on the extentof ulceration in the bladder. In normal rats treated with i.p. saline(saline control), the bladders appeared normal histologically and noulceration of the urothelium was observed. Administration of 200 mg/kgi.p. of cyclophosphamide resulted in ulceration of the bladderepithelium that was between 25 and 50% of the total epithelial area(with a mean of 37%). Administration of KGF-2 Δ33 24 hours prior tocyclophosphamide resulted in a significant reduction in the extent ofulceration (1 mg/kg 0.4% p=0.0128, and 5 mg/kg 5%, p=0.0338%) whencompared to placebo treated animals receiving cyclophosphamide.

[0645]FIG. 55 shows the effects of KGF-2 Δ33 on the thickness of theurinary bladder wall which includes epithelium, smooth muscle layers andthe serosal surface. In groups treated with buffer alone, the thicknessof the bladder wall is approximately 40 μm. Treatment withcyclophosphamide results in a 5 fold increase in bladder wall thicknessto 210 μm. KGF-2 Δ33 pretreatment of cyclophosphamide treated animalsresulted in a significant inhibition of cyclophosphamide enlargement ofthe bladder wall (1 mg/kg 98.6 μm (p=0.007) and at 5 mg/kg 52.3 μm(p<0.0001)) when compared to the cyclophosphamide treatment alone.

[0646] Microscopic Observations

[0647] Prostate Gland: In rats receiving buffer and cyclophosphamide,marked atrophy of the prostatic glands (acini) was observed accompaniedby enlargement of interstitial spaces with remarkable edema whencompared to normals. In addition, epithelial cells lining the prostaticglands were observed to be much shorter and less dense than incorresponding normal prostatic tissue. KGF-2 Δ33 pretreatment at both 1mg/kg and 5 mg/kg displayed a normal histological appearance of theprostatic gland. No increase in the interstitial spaces or edema wasobserved, and the epithelial cells lining the prostatic glands weresimilar in size and density to normal prostatic tissue.

[0648] Conclusion

[0649] The results demonstrate that KGF-2 specifically inducesproliferation of bladder epithelial cells and the epithelial cellslining the prostatic glands. The results also demostrate that KGF-2specifically results in a significant reduction in the extent ofulceration in cyclophosphamide-induced hemorrhagic cystitis.

EXAMPLE 31

[0650] Effect of KGF-2 on the Proliferation of Cells in Normal Rats

[0651] Introduction

[0652] KGF-2, a member of the FGF family, induces proliferation ofnormal human and rat keratinocytes. It has approximately 57% homology toKGF-1 (a member of the FGF family). KGF-1 has been reported to induceproliferation of epithelia of many organs (Housley et al., Keratinocytegrowth factor induces proliferation of hepatocytes and epithelial cellsthroughout the rat gastrointestinal tract. J Clin Invest 94: 1764-1777(1994); Ulich et al., Keratinocyte growth factor is a growth factor fortype II pneumocytes in vivo. J Clin Invest 9.3: 1298-1306 (1994); Ulichet al., Keratinocyte growth factor is a growth factor for mammaryepithelium in vivo. The mammary epithelium of lactating rats isresistant to the proliferative action of keratinocyte growth factor. AmJ Pathol 144:862-868 (1994); Nguyen et al., Expression of keratinocytegrowth factor in embryonic liver of transgenic mice causes changes inepithelial growth and differentiation resulting in polycystic kidneysand other organ malformations. Oncogene 12:2109-2119 (1996); Yi et al.,Keratinocyte growth factor induces pancreatic ductal epithelialproliferation. Am J Pathol 145:80-85 (1994); and Yi et al., Keratinocytegrowth factor causes proliferation of urothelium in vitro. J Urology154:1566-1570 (1995)). We performed similar experiments with KGF-2 todetermine if it induces proliferation of normal epithelia in rats whenadministered systemically using sc and ip routes.

[0653] Methods:

[0654] Male Sprague-Dawley rats, weighing 160-220 g, were obtained fromHarlan Sprague Dawley for these studies. KGF-2 Δ33 (HG03411-E2) wasadministered at a dose of 5 mg/kg/day. Daily ip or sc injections ofKGF-2 Δ33 or recombinant buffer (40 mM sodium acetate +150 mM NaCl at pH6.5) were administered for a period of 1-7 days and the rats weresacrificed the following day (see below). To examine the reversibilityof effects induced with KGF-2 Δ33, additional animals were injected ipdaily for 7 days with KCTF-2 Δ33 or buffer and sacrificed after a 7 daytreatment-free period.

[0655] On the day of sacrifice, rats were injected ip with 100 mg/kg ofBrdU. Two hours later the rats were overdosed with ether and selectedorgans removed. Samples of tissues were fixed in 10% neutral bufferedformalin for 24 hours and paraffin embedded. To detect BrdUincorporation into replicating cells, five micron sections weresubjected to immunohistochemical procedures using a mouse anti-BrdUmonoclonal antibody (Boehringer Mannheim) and the ABC Elite detectionsystem (Vector Laboratories). The sections were lightly counterstainedwith hematoxylin.

[0656] Sections were read by blinded observers. The number ofproliferating cells was counted in 10 random fields per animal at a 1×magnification for the following tissues: liver, pancreas, prostate, andheart. Ten random fields were used also for the lung analysis except theproliferation was quantitated at 20× magnification. Since the kidney hasmany functionally discrete areas, the proliferation was assessed in acoronal cross-section taken through the center of one kidney per animal.To assess the effects of KGF-2 Δ33 in the esophagus and bladder,cross-sections of these tissues were prepared and the number ofproliferating and non-proliferating cells were counted in ten randomfields at a 10× and 20× magnification, respectively. The results areexpressed as the percentage of labeled to unlabeled cells.

[0657] Data are presented as mean±SEM. Statistical analyses (two-tailedunpaired t-test) were performed with the StatView Software Package(Abacus Concepts, Inc., Berkeley, Calif.) and statistical significanceis defined as p<0.05.

[0658] Results

[0659]FIG. 56 shows an overview of the experimental protocol. Sixanimals were used per group. However, during the analysis by the blindedobservers it became clear that occasionally the BrdU injection wasunsuccessful. Before the results were uncoded, the data from 8 rats outof 116 rats (or 7% of the animals) were excluded from the study and theresultant group sizes are shown in the Table below. Group sizes used inthese studies n = Treatment Time ip sc KGF-2 Δ33 1 day 6 5 buffer 1 day6 6 KGF-2 Δ33 2 days 6 4 buffer 2 days 6 6 KGF-2 Δ33 3 days 5 5 buffer 3days 5 5 KGF-2 Δ33 7 days 6 6 buffer 7 days 6 5 KGF-2 Δ33 7 days + 7days treatment-free 6 ND buffer 7 days + 7 days treatment-free 6 ND

[0660] Liver. When administered ip, KGF-2 Δ33 induced a rapidproliferation of hepatocytes (solid squares) (FIG. 57) after 1 injectionand this augmented mitotic activity persisted for three days, returningto normal after 7 days of daily injections. In contrast to the dramaticeffect ip administration of KGF-2 exerted on the liver, when given sc(solid circle, FIG. 57) this growth factor demonstrated minor effects.Proliferation was elevated after one day of treatment but returned tonormal values after two daily injections.

[0661] Pancreas. In contrast to the quickly reversible effects of ipadministered KGF-2 Δ33 on the liver, such injections inducedproliferation of the pancreas which continued over the 14 day studyperiod (solid squares, FIG. 58). Surprisingly, subcutaneousadministration of KGF-2 Δ33 (solid circles) failed to induceproliferation at any time point.

[0662] Kidney and Bladder. KGF-2 Δ33 induced proliferation of renalepithelia when given either by the sc or ip route but the former induceda greater effect. SC administration induced a rapid increase inproliferation (solid circles) that peaked after 2 days which thenreturned to normal after 7 daily treatments (FIG. 59). When KGF-2 Δ33was given ip (solid squares), there was a modest, but significantincrease in proliferation seen at days 2 and 3 only. Intraperitonealinjection of KGF-2 Δ33 also induced proliferation of bladder epithelialcells over the 7 day study period (solid squares, FIG. 52). Subcutaneousadministration elicited a small increase in proliferation but thisfailed to achieve statistical significance (solid circles, FIG. 52).

[0663] Prostate. Both sc and ip administration of KGF-2 Δ33 inducedsignificant proliferation of the prostate (FIG. 53) but this normalizedafter two injections.

[0664] Esophagus. KGF-2 Δ33 given sc or ip elicited an early,short-lived increase in the proliferation of the esophageal cells (1 and2 days, respectively) that rapidly returned to normal (results notshown).

[0665] Other organs. Systemic administration of KGF-2 Δ33 by the ip andsc routes failed to elicit proliferation of the lung epithelia over a 7day dosing period (results not shown).

[0666] Discussion

[0667] When administered in a sc route, we observed stimulation ofnormal epithelial proliferation in some organs (liver, kidney,esophagus. and prostate) but these effects, for the most part, wereshort-lived and all were reversible. The proliferation in these organsreversed even during daily sc administration of KGF-2.

[0668] The route of administration had dramatic effects on the observedproliferation. While daily ip administration increased the rate of liverproliferation over a 3 day period, animals given KGF-2 sc dailyexhibited elevated rates after one day of treatment only. Even moresurprising was the response of the pancreas. When animals were givenKGF-2 ip, the pancreas exhibited a significantly elevated level ofproliferation over the 14 day study period. However, sc administrationof KGF-2 induced no increased mitotic activity in the pancreas.Likewise, ip, but not sc, treatment with K.GF-2 elicited proliferationof the bladder mucosa.

[0669] IP administration of KGF-2 elicited a short-lived, small burst ofproliferation in the kidney that was centered in the region containingcollecting ducts. Daily sc treatment induced a prolonged, exaggeratedproliferation in this area.

EXAMPLE 32

[0670] Effects of KGF-2 Δ33 on Lung Cellular Proliferation FollowingIntratracheal Administration

[0671] The purpose of this example is to show that KGF-2 Δ33 is capableof stimulating lung proliferation in normal rats following intratrachealadministration (administration of KGF-2 Δ33 directly to the lung).

[0672] Methods: Male Lewis rats (220-270 g), (n=5/treatment group) wereused in these studies. KGF-2 Δ33 or placebo (40 mM sodium acetate +150mM NaCl at pH 6.5) was administered intratracheally at doses of 1 and 5mg/kg in a volume of 0.6 mls followed by 3 mls of air. Treatments wereadministered on stay 1 and day 2 of the experimental protocol.

[0673] On day 3, the day of sacrifice, rats were injected ip with 100mg/kg of BrdU. Two hours later the rats were killed by C02 asphyxiation.Lungs were inflated with 10% buffered formalin via intratrachealcatheter, and saggital sections of lung were paraffin embedded. Todetect BrdU incorporation into replicating cells, five micron sectionswere subjected to immunohistochemical procedures using a mouse anti-BrdUmonoclonal antibody and the ABC Elite detection system. The sectionswere lightly counterstained with hematoxylin.

[0674] Sections were read by two blinded observers. The number ofproliferating cells was counted in 10 random fields per section at a 20× magnification. The results are expressed as the number of BrdUpositive cells per field. Data are presented as mean +SEM. Statisticalanalyses (unpaired t-test) were performed with the Instat v2.0.1 andstatistical significance is defined as p<0.05.

[0675] Results: Intratracheal injection of KGF-2 Δ33 at 1 and 5 mg/kgresulted in an increase in proliferation of lung epithelial cells asshown in FIG. 60. KGF-2 Δ33 treatment resulted in statisticallysignificant increases in the number of BrdU positive cells/field at 1mg/kg 23.4 cells/field (p=0.0002) and at 5 mg/kg 10.3 cells/field(p0.0003) relative to buffer controls of 1.58 cells per field.

[0676] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0677] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

[0678] The entire disclosure of all publications (including patents,patent applications, journal articles, laboratory manuals, books, orother documents) cited herein are hereby incorporated by reference.

1 148 627 base pairs nucleic acid double both DNA (genomic) CDS 1..624 1ATG TGG AAA TGG ATA CTG ACA CAT TGT GCC TCA GCC TTT CCC CAC CTG 48 MetTrp Lys Trp Ile Leu Thr His Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15CCC GGC TGC TGC TGC TGC TGC TTT TTG TTG CTG TTC TTG GTG TCT TCC 96 ProGly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 GTCCCT GTC ACC TGC CAA GCC CTT GGT CAG GAC ATG GTG TCA CCA GAG 144 Val ProVal Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 GCC ACCAAC TCT TCT TCC TCC TCC TTC TCC TCT CCT TCC AGC GCG GGA 192 Ala Thr AsnSer Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly 50 55 60 AGG CAT GTGCGG AGC TAC AAT CAC CTT CAA GGA GAT GTC CGC TGG AGA 240 Arg His Val ArgSer Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 AAG CTA TTCTCT TTC ACC AAG TAC TTT CTC AAG ATT GAG AAG AAC GGG 288 Lys Leu Phe SerPhe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly 85 90 95 AAG GTC AGC GGGACC AAG AAG GAG AAC TGC CCG TAC AGC ATC CTG GAG 336 Lys Val Ser Gly ThrLys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu 100 105 110 ATA ACA TCA GTAGAA ATC GGA GTT GTT GCC GTC AAA GCC ATT AAC AGC 384 Ile Thr Ser Val GluIle Gly Val Val Ala Val Lys Ala Ile Asn Ser 115 120 125 AAC TAT TAC TTAGCC ATG AAC AAG AAG GGG AAA CTC TAT GGC TCA AAA 432 Asn Tyr Tyr Leu AlaMet Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 GAA TTT AAC AATGAC TGT AAG CTG AAG GAG AGG ATA GAG GAA AAT GGA 480 Glu Phe Asn Asn AspCys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly 145 150 155 160 TAC AAT ACCTAT GCA TCA TTT AAC TGG CAG CAT AAT GGG AGG CAA ATG 528 Tyr Asn Thr TyrAla Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175 TAT GTG GCATTG AAT GGA AAA GGA GCT CCA AGG AGA GGA CAG AAA ACA 576 Tyr Val Ala LeuAsn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr 180 185 190 CGA AGG AAAAAC ACC TCT GCT CAC TTT CTT CCA ATG GTG GTA CAC TCA 624 Arg Arg Lys AsnThr Ser Ala His Phe Leu Pro Met Val Val His Ser 195 200 205 TAG 627 208amino acids amino acid linear protein 2 Met Trp Lys Trp Ile Leu Thr HisCys Ala Ser Ala Phe Pro His Leu 1 5 10 15 Pro Gly Cys Cys Cys Cys CysPhe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 Val Pro Val Thr Cys Gln AlaLeu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 Ala Thr Asn Ser Ser Ser SerSer Phe Ser Ser Pro Ser Ser Ala Gly 50 55 60 Arg His Val Arg Ser Tyr AsnHis Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 Lys Leu Phe Ser Phe ThrLys Tyr Phe Leu Lys Ile Glu Lys Asn Gly 85 90 95 Lys Val Ser Gly Thr LysLys Glu Asn Cys Pro Tyr Ser Ile Leu Glu 100 105 110 Ile Thr Ser Val GluIle Gly Val Val Ala Val Lys Ala Ile Asn Ser 115 120 125 Asn Tyr Tyr LeuAla Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 Glu Phe AsnAsn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly 145 150 155 160 TyrAsn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr 180 185190 Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 195200 205 36 base pairs nucleic acid single linear cDNA 3 CCCCACATGTGGAAATGGAT ACTGACACAT TGTGCC 36 35 base pairs nucleic acid single linearcDNA 4 CCCAAGCTTC CACAAACGTT GCCTTCCTCT ATGAG 35 36 base pairs nucleicacid single linear cDNA 5 CATGCCATGG CGTGCCAAGC CCTTGGTCAG GACATG 36 35base pairs nucleic acid single linear cDNA 6 CCCAAGCTTC CACAAACGTTGCCTTCCTCT ATGAG 35 35 base pairs nucleic acid single linear cDNA 7GCGGGATCCG CCATCATGTG GAAATGGATA CTCAC 35 27 base pairs nucleic acidsingle linear cDNA 8 GCGCGGTACC ACAAACGTTG CCTTCCT 27 40 base pairsnucleic acid single linear cDNA 9 TAACGAGGAT CCGCCATCAT GTGGAAATGGATACTGACAC 40 38 base pairs nucleic acid single linear cDNA 10TAAGCACTCG AGTGAGTGTA CCACCATTGG AAGAAATG 38 54 base pairs nucleic acidsingle linear cDNA 11 ATTAACCCTC ACTAAAGGGA GGCCATGTGG AAATGGATACTGACACATTG TGCC 54 35 base pairs nucleic acid single linear cDNA 12CCCAAGCTTC CACAAACGTT GCCTTCCTCT ATGAG 35 206 amino acids amino acid NotRelevant Not Relevant protein 13 Met Ser Gly Pro Gly Thr Ala Ala Val AlaLeu Leu Pro Ala Val Leu 1 5 10 15 Leu Ala Leu Leu Ala Pro Trp Ala GlyArg Gly Gly Ala Ala Ala Pro 20 25 30 Thr Ala Pro Asn Gly Thr Leu Glu AlaGlu Leu Glu Arg Arg Trp Glu 35 40 45 Ser Leu Val Ala Leu Ser Leu Ala ArgLeu Pro Val Ala Ala Gln Pro 50 55 60 Lys Glu Ala Ala Val Gln Ser Gly AlaGly Asp Tyr Leu Leu Gly Ile 65 70 75 80 Lys Arg Leu Arg Arg Leu Tyr CysAsn Val Gly Ile Gly Phe His Leu 85 90 95 Gln Ala Leu Pro Asp Gly Arg IleGly Gly Ala His Ala Asp Thr Arg 100 105 110 Asp Ser Leu Leu Glu Leu SerPro Val Glu Arg Gly Val Val Ser Ile 115 120 125 Phe Gly Val Ala Ser ArgPhe Phe Val Ala Met Ser Ser Lys Gly Lys 130 135 140 Leu Tyr Gly Ser ProPhe Phe Thr Asp Glu Cys Thr Phe Lys Glu Ile 145 150 155 160 Leu Leu ProAsn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly 165 170 175 Met PheIle Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg 180 185 190 ValSer Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu 195 200 205 198amino acids amino acid Not Relevant Not Relevant protein 14 Met Ser ArgGly Ala Gly Arg Leu Gln Gly Thr Leu Trp Ala Leu Val 1 5 10 15 Phe LeuGly Ile Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Thr 20 25 30 Arg AlaAsn Asn Thr Leu Leu Asp Ser Arg Gly Trp Gly Thr Leu Leu 35 40 45 Ser ArgSer Arg Ala Gly Leu Ala Gly Glu Ile Ala Gly Val Asn Trp 50 55 60 Glu SerGly Tyr Leu Val Gly Ile Lys Arg Gln Arg Arg Leu Tyr Cys 65 70 75 80 AsnVal Gly Ile Gly Phe His Leu Gln Val Leu Pro Asp Gly Arg Ile 85 90 95 SerGly Thr His Glu Glu Asn Pro Tyr Ser Leu Leu Glu Ile Ser Thr 100 105 110Val Glu Arg Gly Val Val Ser Leu Phe Gly Val Arg Ser Ala Leu Phe 115 120125 Val Ala Met Asn Ser Lys Gly Arg Leu Tyr Ala Thr Pro Ser Phe Gln 130135 140 Glu Glu Cys Lys Phe Arg Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala145 150 155 160 Tyr Glu Ser Asp Leu Tyr Gln Gly Thr Tyr Ile Ala Leu SerLys Tyr 165 170 175 Gly Arg Val Lys Arg Gly Ser Lys Val Ser Pro Ile MetThr Val Thr 180 185 190 His Phe Leu Pro Arg Ile 195 268 amino acidsamino acid Not Relevant Not Relevant protein 15 Met Ser Leu Ser Phe LeuLeu Leu Leu Phe Phe Ser His Leu Ile Leu 1 5 10 15 Ser Ala Trp Ala HisGly Glu Lys Arg Leu Ala Pro Lys Gly Gln Pro 20 25 30 Gly Pro Ala Ala ThrAsp Arg Asn Pro Arg Gly Ser Ser Ser Arg Gln 35 40 45 Ser Ser Ser Ser AlaMet Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala 50 55 60 Ala Ser Leu Gly SerGln Gly Ser Gly Leu Glu Gln Ser Ser Phe Gln 65 70 75 80 Trp Ser Pro SerGly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly 85 90 95 Ile Gly Phe HisLeu Gln Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser 100 105 110 His Glu AlaAsn Met Leu Ser Val Leu Glu Ile Phe Ala Val Ser Gln 115 120 125 Gly IleVal Gly Ile Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met 130 135 140 SerLys Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys 145 150 155160 Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser 165170 175 Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu180 185 190 Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val LysPro 195 200 205 Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys Gln SerGlu Gln 210 215 220 Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys LysAsn Pro Pro 225 230 235 240 Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser AlaPro Arg Lys Asn Thr 245 250 255 Asn Ser Val Lys Tyr Arg Leu Lys Phe ArgPhe Gly 260 265 155 amino acids amino acid Not Relevant Not Relevantprotein 16 Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu LysPhe 1 5 10 15 Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu TyrCys Ser 20 25 30 Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr ValAsp Gly 35 40 45 Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu SerAla Glu 50 55 60 Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly GlnTyr Leu 65 70 75 80 Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln ThrPro Asn Glu 85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His TyrAsn Thr Tyr 100 105 110 Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe ValGly Leu Lys Lys 115 120 125 Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr HisTyr Gly Gln Lys Ala 130 135 140 Ile Leu Phe Leu Pro Leu Pro Val Ser SerAsp 145 150 155 155 amino acids amino acid Not Relevant Not Relevantprotein 17 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu AspGly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro LysArg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro AspGly Arg 35 40 45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys LeuGln Leu 50 55 60 Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val CysAla Asn 65 70 75 80 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu AlaSer Lys Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu SerAsn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp TyrVal Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys ThrGly Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met Ser Ala LysSer 145 150 155 208 amino acids amino acid Not Relevant Not Relevantprotein 18 Met Ala Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln AspAla 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser ProVal Leu 20 25 30 Leu Ser Asp His Leu Gly Gln Ser Glu Ala Gly Gly Leu ProArg Gly 35 40 45 Pro Ala Val Thr Asp Leu Asp His Leu Lys Gly Ile Leu ArgArg Arg 50 55 60 Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe ProAsn Gly 65 70 75 80 Thr Ile Gln Gly Thr Arg Lys Asp His Ser Arg Phe GlyIle Leu Glu 85 90 95 Phe Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg GlyVal Asp Ser 100 105 110 Gly Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu LeuTyr Gly Ser Glu 115 120 125 Lys Leu Thr Gln Glu Cys Val Phe Arg Glu GlnPhe Glu Glu Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser Ser Asn Leu Tyr LysHis Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr Val Ala Leu Asn LysAsp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr Lys Arg His Gln LysPhe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp Pro Asp Lys Val ProGlu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205 194 amino acidsamino acid Not Relevant Not Relevant protein 19 Met His Lys Trp Ile LeuThr Trp Ile Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15 Ser Cys Phe His IleIle Cys Leu Val Gly Thr Ile Ser Leu Ala Cys 20 25 30 Asn Asp Met Thr ProGlu Gln Met Ala Thr Asn Val Asn Cys Ser Ser 35 40 45 Pro Glu Arg His ThrArg Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile 50 55 60 Arg Val Arg Arg LeuPhe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp 65 70 75 80 Lys Arg Gly LysVal Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn 85 90 95 Ile Met Glu IleArg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly 100 105 110 Val Glu SerGlu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115 120 125 Ala LysLys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu 130 135 140 GluAsn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly 145 150 155160 Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly 165170 175 Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala180 185 190 Ile Thr 208 amino acids amino acid Not Relevant Not Relevantprotein 20 Met Trp Lys Trp Ile Leu Thr His Cys Ala Ser Ala Phe Pro HisLeu 1 5 10 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu Leu Phe Leu ValSer Ser 20 25 30 Val Pro Val Thr Cys Gln Ala Leu Gly Gln Asp Met Val SerPro Glu 35 40 45 Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser SerAla Gly 50 55 60 Arg His Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val ArgTrp Arg 65 70 75 80 Lys Leu Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile GluLys Asn Gly 85 90 95 Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr SerIle Leu Glu 100 105 110 Ile Thr Ser Val Glu Ile Gly Val Val Ala Val LysAla Ile Asn Ser 115 120 125 Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly LysLeu Tyr Gly Ser Lys 130 135 140 Glu Phe Asn Asn Asp Cys Lys Leu Lys GluArg Ile Glu Glu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr Ala Ser Phe AsnTrp Gln His Asn Gly Arg Gln Met 165 170 175 Tyr Val Ala Leu Asn Gly LysGly Ala Pro Arg Arg Gly Gln Lys Thr 180 185 190 Arg Arg Lys Asn Thr SerAla His Phe Leu Pro Met Val Val His Ser 195 200 205 239 amino acidsamino acid Not Relevant Not Relevant protein 21 Met Gly Leu Ile Trp LeuLeu Leu Leu Ser Leu Leu Glu Pro Gly Trp 1 5 10 15 Pro Ala Ala Gly ProGly Ala Arg Leu Arg Arg Asp Ala Gly Gly Arg 20 25 30 Gly Gly Val Tyr GluHis Leu Gly Gly Ala Pro Arg Arg Arg Lys Leu 35 40 45 Tyr Cys Ala Thr LysTyr His Leu Gln Leu His Pro Ser Gly Arg Val 50 55 60 Asn Gly Ser Leu GluAsn Ser Ala Tyr Ser Ile Leu Glu Ile Thr Ala 65 70 75 80 Val Glu Val GlyIle Val Ala Ile Arg Gly Leu Phe Ser Gly Arg Tyr 85 90 95 Leu Ala Met AsnLys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr Ser 100 105 110 Ala Glu CysGlu Phe Val Glu Arg Ile His Glu Leu Gly Tyr Asn Thr 115 120 125 Tyr AlaSer Arg Leu Tyr Arg Thr Val Ser Ser Thr Pro Gly Ala Arg 130 135 140 ArgGln Pro Ser Ala Glu Arg Leu Trp Tyr Val Ser Val Asn Gly Lys 145 150 155160 Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gln Lys Ser Ser 165170 175 Leu Phe Leu Pro Arg Val Leu Asp His Arg Asp His Glu Met Val Arg180 185 190 Gln Leu Gln Ser Gly Leu Pro Arg Pro Pro Gly Lys Gly Val GlnPro 195 200 205 Arg Arg Arg Arg Gln Lys Gln Ser Pro Asp Asn Leu Glu ProSer His 210 215 220 Val Gln Ala Ser Arg Leu Gly Ser Gln Leu Glu Ala SerAla His 225 230 235 268 amino acids amino acid Not Relevant Not Relevantprotein 22 Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu Leu His LeuLeu 1 5 10 15 Val Leu Cys Leu Gln Ala Gln Val Arg Ser Ala Ala Gln LysArg Gly 20 25 30 Pro Gly Ala Gly Asn Pro Ala Asp Thr Leu Gly Gln Gly HisGlu Asp 35 40 45 Arg Pro Phe Gly Gln Arg Ser Arg Ala Gly Lys Asn Phe ThrAsn Pro 50 55 60 Ala Pro Asn Tyr Pro Glu Glu Gly Ser Lys Glu Gln Arg AspSer Val 65 70 75 80 Leu Pro Lys Val Thr Gln Arg His Val Arg Glu Gln SerLeu Val Thr 85 90 95 Asp Gln Leu Ser Arg Arg Leu Ile Arg Thr Tyr Gln LeuTyr Ser Arg 100 105 110 Thr Ser Gly Lys His Val Gln Val Leu Ala Asn LysArg Ile Asn Ala 115 120 125 Met Ala Glu Asp Gly Asp Pro Phe Ala Lys LeuIle Val Glu Thr Asp 130 135 140 Thr Phe Gly Ser Arg Val Arg Val Arg GlyAla Glu Thr Gly Leu Tyr 145 150 155 160 Ile Cys Met Asn Lys Lys Gly LysLeu Ile Ala Lys Ser Asn Gly Lys 165 170 175 Gly Lys Asp Cys Val Phe ThrGlu Ile Val Leu Glu Asn Asn Tyr Thr 180 185 190 Ala Leu Gln Asn Ala LysTyr Glu Gly Trp Tyr Met Ala Phe Thr Arg 195 200 205 Lys Gly Arg Pro ArgLys Gly Ser Lys Thr Arg Gln His Gln Arg Glu 210 215 220 Val His Phe MetLys Arg Leu Pro Arg Gly His His Thr Thr Glu Gln 225 230 235 240 Ser LeuArg Phe Glu Phe Leu Asn Tyr Pro Pro Phe Thr Arg Ser Leu 245 250 255 ArgGly Ser Gln Arg Thr Trp Ala Pro Glu Pro Arg 260 265 4177 base pairsnucleic acid double both DNA (genomic) CDS 593..1216 23 GGAATTCCGGGAAGAGAGGG AAGAAAACAA CGGCGACTGG GCAGCTGCCT CCACTTCTGA 60 CAACTCCAAAGGGATATACT TGTAGAAGTG GCTCGCAGGC TGGGGCTCCG CAGAGAGAGA 120 CCAGAAGGTGCCAACCGCAG AGGGGTGCAG ATATCTCCCC CTATTCCCCA CCCCACCTCC 180 CTTGGGTTTTGTTCACCGTG CTGTCATCTG TTTTTCAGAC CTTTTTGGCA TCTAACATGG 240 TGAAGAAAGGAGTAAAGAAG AGAACAAAGT AACTCCTGGG GGAGCGAAGA GCGCTGGTGA 300 CCAACACCACCAACGCCACC ACCAGCTCCT GCTGCTGCGG CCACCCACGT CCACCATTTA 360 CCGGGAGGCTCCAGAGGCGT AGGCAGCGGA TCCGAGAAAG GAGCGAGGGG AGTCAGCCGG 420 CTTTTCCGAGGAGTTATGGA TGTTGGTGCA TTCACTTCTG GCCAGATCCG CGCCCAGAGG 480 GAGCTAACCAGCAGCCACCA CCTCGAGCTC TCTCCTTGCC TTGCATCGGG TCTTACCCTT 540 CCAGTATGTTCCTTCTGATG AGACAATTTC CAGTGCCGAG AGTTTCAGTA CA ATG 595 Met 1 TGG AAA TGGATA CTG ACA CAT TGT GCC TCA GCC TTT CCC CAC CTG CCC 643 Trp Lys Trp IleLeu Thr His Cys Ala Ser Ala Phe Pro His Leu Pro 5 10 15 GGC TGC TGC TGCTGC TGC TTT TTG TTG CTG TTC TTG GTG TCT TCC GTC 691 Gly Cys Cys Cys CysCys Phe Leu Leu Leu Phe Leu Val Ser Ser Val 20 25 30 CCT GTC ACC TGC CAAGCC CTT GGT CAG GAC ATG GTG TCA CCA GAG GCC 739 Pro Val Thr Cys Gln AlaLeu Gly Gln Asp Met Val Ser Pro Glu Ala 35 40 45 ACC AAC TCT TCT TCC TCCTCC TTC TCC TCT CCT TCC AGC GCG GGA AGG 787 Thr Asn Ser Ser Ser Ser SerPhe Ser Ser Pro Ser Ser Ala Gly Arg 50 55 60 65 CAT GTG CGG AGC TAC AATCAC CTT CAA GGA GAT GTC CGC TGG AGA AAG 835 His Val Arg Ser Tyr Asn HisLeu Gln Gly Asp Val Arg Trp Arg Lys 70 75 80 CTA TTC TCT TTC ACC AAG TACTTT CTC AAG ATT GAG AAG AAC GGG AAG 883 Leu Phe Ser Phe Thr Lys Tyr PheLeu Lys Ile Glu Lys Asn Gly Lys 85 90 95 GTC AGC GGG ACC AAG AAG GAG AACTGC CCG TAC AGC ATC CTG GAG ATA 931 Val Ser Gly Thr Lys Lys Glu Asn CysPro Tyr Ser Ile Leu Glu Ile 100 105 110 ACA TCA GTA GAA ATC GGA GTT GTTGCC GTC AAA GCC ATT AAC AGC AAC 979 Thr Ser Val Glu Ile Gly Val Val AlaVal Lys Ala Ile Asn Ser Asn 115 120 125 TAT TAC TTA GCC ATG AAC AAG AAGGGG AAA CTC TAT GGC TCA AAA GAA 1027 Tyr Tyr Leu Ala Met Asn Lys Lys GlyLys Leu Tyr Gly Ser Lys Glu 130 135 140 145 TTT AAC AAT GAC TGT AAG CTGAAG GAG AGG ATA GAG GAA AAT GGA TAC 1075 Phe Asn Asn Asp Cys Lys Leu LysGlu Arg Ile Glu Glu Asn Gly Tyr 150 155 160 AAT ACC TAT GCA TCA TTT AACTGG CAG CAT AAT GGG AGG CAA ATG TAT 1123 Asn Thr Tyr Ala Ser Phe Asn TrpGln His Asn Gly Arg Gln Met Tyr 165 170 175 GTG GCA TTG AAT GGA AAA GGAGCT CCA AGG AGA GGA CAG AAA ACA CGA 1171 Val Ala Leu Asn Gly Lys Gly AlaPro Arg Arg Gly Gln Lys Thr Arg 180 185 190 AGG AAA AAC ACC TCT GCT CACTTT CTT CCA ATG GTG GTA CAC TCA 1216 Arg Lys Asn Thr Ser Ala His Phe LeuPro Met Val Val His Ser 195 200 205 TAGAGGAAGG CAACGTTTGT GGATGCAGTAAAACCAATGG CTCTTTTGCC AAGAATAGTG 1276 GATATTCTTC ATGAAGACAG TAGATTGAAAGGCAAAGACA CGTTGCAGAT GTCTGCTTGC 1336 TTAAAAGAAA GCCAGCCTTT GAAGGTTTTTGTATTCACTG CTGACATATG ATGTTCTTTT 1396 AATTAGTTCT GTGTCATGTC TTATAATCAAGATATAGGCA GATCGAATGG GATAGAAGTT 1456 ATTCCCAAGT GAAAAACATT GTGGCTGGGTTTTTTGTTGT TGTTGTCAAG TTTTTGTTTT 1516 TAAACCTCTG AGATAGAACT TAAAGGACATAGAACAATCT GTTGAAAGAA CGATCTTCGG 1576 GAAAGTTATT TATGGAATAC GAACTCATATCAAAGACTTC ATTGCTCATT CAAGCCTAAT 1636 GAATCAATGA ACAGTAATAC GTGCAAGCATTTACTGGAAA GCACTTGGGT CATATCATAT 1696 GCACAACCAA AGGAGTTCTG GATGTGGTCTCATGGAATAA TTGAATAGAA TTTAAAAATA 1756 TAAACATGTT AGTGTGAAAC TGTTCTAACAATACAAATAG TATGGTATGC TTGTGCATTC 1816 TGCCTTCATC CCTTTCTATT TCTTTCTAAGTTATTTATTT AATAGGATGT TAAATATCTT 1876 TTGGGGTTTT AAAGAGTATC TCAGCAGCTGTCTTCTGATT TATCTTTTCT TTTTATTCAG 1936 CACACCACAT GCATGTTCAC GACAAAGTGTTTTTAAAACT TGGCGAACAC TTCAAAAATA 1996 GGAGTTGGGA TTAGGGAAGC AGTATGAGTGCCCGTGTGCT ATCAGTTGAC TTAATTTGCA 2056 CTTCTGCAGT AATAACCATC AACAATAAATATGGCAATGC TGTGCCATGG CTTGAGTGAG 2116 AGATGTCTGC TATCATTTGA AAACATATATTACTCTCGAG GCTTCCTGTC TCAAGAAATA 2176 GACCAGAAGG CCAAATTCTT CTCTTTCAATACATCAGTTT GCCTCCAAGA ATATACTAAA 2236 AAAAGGAAAA TTAATTGCTA AATACATTTAAATAGCCTAG CCTCATTATT TACTCATGAT 2296 TTCTTGCCAA ATGTCATGGC GGTAAAGAGGCTGTCCACAT CTCTAAAAAC CCTCTGTAAA 2356 TTCCACATAA TGCATCTTTC CCAAAGGAACTATAAAGAAT TTGGTATGAA GCGCAACTCT 2416 CCCAGGGGCT TAAACTGAGC AAATCAAATATATACTGGTA TATGTGTAAC CATATACAAA 2476 AACCTGTTCT AGCTGTATGA TCTAGTCTTTACAAAACCAA ATAAAACTTG TTTTCTGTAA 2536 ATTTAAAGAG CTTTACAAGG TTCCATAATGTAACCATATC AAAATTCATT TTGTTAGAGC 2596 ACGTATAGAA AAGAGTACAT AAGAGTTTACCAATCATCAT CACATTGTAT TCCACTAAAT 2656 AAATACATAA GCCTTATTTG CAGTGTCTGTAGTGATTTTA AAAATGTAGA AAAATACTAT 2716 TTGTTCTAAA TACTTTTAAG CAATAACTATAATAGTATAT TGATGCTGCA GTTTTATCTT 2776 CATATTTCTT GTTTTGAAAA AGCATTTTATTGTTTGGACA CAGTATTTTG GTACAAAAAA 2836 AAAGACTCAC TAAATGTGTC TTACTAAAGTTTAACCTTTG GAAATGCTGG CGTTCTGTGA 2896 TTCTCCAACA AACTTATTTG TGTCAATACTTAACCAGCAC TTCCAGTTAA TCTGTTATTT 2956 TTAAAAATTG CTTTATTAAG AAATTTTTTGTATAATCCCA TAAAAGGTCA TATTTTTCCC 3016 ATTCTTCAAA AAAACTGTAT TTCAGAAGAAACACATTTGA GGCACTGTCT TTTGGCTTAT 3076 AGTTTAAATT GCATTTCATC ATACTTTGCTTCCAACTTGC TTTTTGGCAA ATGAGATTAT 3136 AAAAATGTTT AATTTTTGTG GTTGGAATCTGGATGTTAAA ATTTAATTGG TAACTCAGTC 3196 TGTGAGCTAT AATGTAATGC ATTCCTATCCAAACTAGGTA TCTTTTTTTC CTTTATGTTG 3256 AAATAATAAT GGCACCTGAC ACATAGACATAGACCACCCA CAACCTAAAT TAAATGTTTG 3316 GTAAGACAAA TACACATTGG ATGACCACAGTAACAGCAAA CAGGGCACAA ACTGGATTCT 3376 TATTTCACAT AGACATTTAG ATTACTAAAGAGGGCTATGT GTAAACAGTC ATCATTATAG 3436 TACTCAAGAC ACTAAAACAG CTTCTAGCCAAATATATTAA AGCTTGCAGA GGCCAAAAAT 3496 AGAAAACATC TCCCCTGTCT CTCCCACATTTCCCTCACAG AAAGACAAAA AACCTGCCTG 3556 GTGCAGTAGC TCACACCTGT AATCCCAGCAGTTTGGGAGA CTGTGGGAAG ATGGCTTGAG 3616 TCCAGGAGTT CTAGACAGGC CTGAGAAACCTAGTGAGACA TCCTTCTCTT AAACAAAACA 3676 AAACAAAACA AATGTAGCCA TGCGTGGTGGCATATACCTG TGGTCCCAAC TACTCAGGAG 3736 GCTGAAACGG AAGGATCTCT TGGGCCCCAGGAGTTTGAGG CTGCAGTGAG CTATAATCTT 3796 GCCATTGCAC TCCAGCCTGG GTGAAAAAGAGCCAGAAAGA AAGGAAAGAG AGAAAAGAGA 3856 AAAGAAAGAG AGAAAAGACA GAAAGACAGGAAGGAAGGAA GGAAGGAAGG AAGGAAGGAA 3916 GGAAGCAAGG AAAGAAGGAA GGAAGGAAAGAAGGGAGGGA AGGAAGGAGA GAGAAAGAAA 3976 GATTGTTTGG TAAGGAGTAA TGACATTCTCTTGCATTTAA AAGTGGCATA TTTGCTTGAA 4036 ATGGAAATAG AATTCTGGTC CCTTTTGCAACTACTGAAGA AAAAAAAAAG CAGTTTCAGC 4096 CCTGAATGTT GTAGATTTGA AAAAAAAAAAAAAAAAACTC GAGGGGGGGC CCGTACCCAA 4156 TTCGCCCTAT AGTGAGTCGT A 4177 208amino acids amino acid linear protein 24 Met Trp Lys Trp Ile Leu Thr HisCys Ala Ser Ala Phe Pro His Leu 1 5 10 15 Pro Gly Cys Cys Cys Cys CysPhe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 Val Pro Val Thr Cys Gln AlaLeu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 Ala Thr Asn Ser Ser Ser SerSer Phe Ser Ser Pro Ser Ser Ala Gly 50 55 60 Arg His Val Arg Ser Tyr AsnHis Leu Gln Gly Asp Val Arg Trp Arg 65 70 75 80 Lys Leu Phe Ser Phe ThrLys Tyr Phe Leu Lys Ile Glu Lys Asn Gly 85 90 95 Lys Val Ser Gly Thr LysLys Glu Asn Cys Pro Tyr Ser Ile Leu Glu 100 105 110 Ile Thr Ser Val GluIle Gly Val Val Ala Val Lys Ala Ile Asn Ser 115 120 125 Asn Tyr Tyr LeuAla Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 Glu Phe AsnAsn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly 145 150 155 160 TyrAsn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr 180 185190 Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 195200 205 31 amino acids amino acid Not Relevant Not Relevant peptide 25Gly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser Ser Ser Ser 1 5 1015 Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr Asn 20 25 3019 amino acids amino acid Not Relevant Not Relevant peptide 26 Lys IleGlu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys 1 5 10 15 ProTyr Ser 30 amino acids amino acid Not Relevant Not Relevant peptide 27Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys 1 5 1015 Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr Tyr 20 25 30 19amino acids amino acid Not Relevant Not Relevant peptide 28 Asn Gly LysGly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn 1 5 10 15 Thr SerAla 555 base pairs nucleic acid double linear DNA (genomic) CDS 1..55329 ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC GGA TCC TGC CAG GCT CTG 48Met Arg Gly Ser His His His His His His Gly Ser Cys Gln Ala Leu 1 5 1015 GGT CAG GAC ATG GTT TCT CCG GAA GCT ACC AAC TCT TCC TCT TCC TCT 96Gly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser Ser Ser Ser 20 25 30TTC TCT TCC CCG TCT TCC GCT GGT CGT CAC GTT CGT TCT TAC AAC CAC 144 PheSer Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr Asn His 35 40 45 CTGCAG GGT GAC GTT CGT TGG CGT AAA CTG TTC TCT TTC ACC AAA TAC 192 Leu GlnGly Asp Val Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr 50 55 60 TTC CTGAAA ATC GAA AAA AAC GGT AAA GTT TCT GGG ACC AAG AAG GAG 240 Phe Leu LysIle Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu 65 70 75 80 AAC TGCCCG TAC AGC ATC CTG GAG ATA ACA TCA GTA GAA ATC GGA GTT 288 Asn Cys ProTyr Ser Ile Leu Glu Ile Thr Ser Val Glu Ile Gly Val 85 90 95 GTT GCC GTCAAA GCC ATT AAC AGC AAC TAT TAC TTA GCC ATG AAC AAG 336 Val Ala Val LysAla Ile Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys 100 105 110 AAG GGG AAACTC TAT GGC TCA AAA GAA TTT AAC AAT GAC TGT AAG CTG 384 Lys Gly Lys LeuTyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu 115 120 125 AAG GAG AGGATA GAG GAA AAT GGA TAC AAT ACC TAT GCA TCA TTT AAC 432 Lys Glu Arg IleGlu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn 130 135 140 TGG CAG CATAAT GGG AGG CAA ATG TAT GTG GCA TTG AAT GGA AAA GGA 480 Trp Gln His AsnGly Arg Gln Met Tyr Val Ala Leu Asn Gly Lys Gly 145 150 155 160 GCT CCAAGG AGA GGA CAG AAA ACA CGA AGG AAA AAC ACC TCT GCT CAC 528 Ala Pro ArgArg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Ala His 165 170 175 TTT CTTCCA ATG GTG GTA CAC TCA TAG 555 Phe Leu Pro Met Val Val His Ser 180 184amino acids amino acid linear protein 30 Met Arg Gly Ser His His His HisHis His Gly Ser Cys Gln Ala Leu 1 5 10 15 Gly Gln Asp Met Val Ser ProGlu Ala Thr Asn Ser Ser Ser Ser Ser 20 25 30 Phe Ser Ser Pro Ser Ser AlaGly Arg His Val Arg Ser Tyr Asn His 35 40 45 Leu Gln Gly Asp Val Arg TrpArg Lys Leu Phe Ser Phe Thr Lys Tyr 50 55 60 Phe Leu Lys Ile Glu Lys AsnGly Lys Val Ser Gly Thr Lys Lys Glu 65 70 75 80 Asn Cys Pro Tyr Ser IleLeu Glu Ile Thr Ser Val Glu Ile Gly Val 85 90 95 Val Ala Val Lys Ala IleAsn Ser Asn Tyr Tyr Leu Ala Met Asn Lys 100 105 110 Lys Gly Lys Leu TyrGly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu 115 120 125 Lys Glu Arg IleGlu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn 130 135 140 Trp Gln HisAsn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly Lys Gly 145 150 155 160 AlaPro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Ala His 165 170 175Phe Leu Pro Met Val Val His Ser 180 84 base pairs nucleic acid singlelinear cDNA 31 ATGTGGAAAT GGATACTGAC CCACTGCGCT TCTGCTTTCC CGCACCTGCCGGGTTGCTGC 60 TGCTGCTGCT TCCTGCTGCT GTTC 84 82 base pairs nucleic acidsingle linear cDNA 32 CCGGAGAAAC CATGTCCTGA CCCAGAGCCT GGCAGGTAACCGGAACAGAA GAAACCAGGA 60 ACAGCAGCAG GAAGCAGCAG CA 82 80 base pairsnucleic acid single linear cDNA 33 GGGTCAGGAC ATGGTTTCTC CGGAAGCTACCAACTCTTCT TCTTCTTCTT TCTCTTCTCC 60 GTCTTCTGCT GGTCGTCACG 80 81 basepairs nucleic acid single linear cDNA 34 GGTGAAAGAG AACAGTTTACGCCAACGAAC GTCACCCTGC AGGTGGTTGT AAGAACGAAC 60 GTGACGACCA GCAGAAGACG G81 75 base pairs nucleic acid single linear cDNA 35 CGTTGGCGTAAACTGTTCTC TTTCACCAAA TACTTCCTGA AAATCGAAAA AAACGGTAAA 60 GTTTCTGGGACCAAA 75 39 base pairs nucleic acid single linear cDNA 36 TTTGGTCCCAGAAACTTTAC CGTTTTTTTC GATTTTCAG 39 36 base pairs nucleic acid singlelinear cDNA 37 AAAGGATCCA TGTGGAAATG GATACTGACC CACTGC 36 627 base pairsnucleic acid double both DNA (genomic) CDS 1..624 38 ATG TGG AAA TGG ATACTG ACC CAC TGC GCT TCT GCT TTC CCG CAC CTG 48 Met Trp Lys Trp Ile LeuThr His Cys Ala Ser Ala Phe Pro His Leu 1 5 10 15 CCG GGT TGC TGC TGCTGC TGC TTC CTG CTG CTG TTC CTG GTT TCT TCT 96 Pro Gly Cys Cys Cys CysCys Phe Leu Leu Leu Phe Leu Val Ser Ser 20 25 30 GTT CCG GTT ACC TGC CAGGCT CTG GGT CAG GAC ATG GTT TCT CCG GAA 144 Val Pro Val Thr Cys Gln AlaLeu Gly Gln Asp Met Val Ser Pro Glu 35 40 45 GCT ACC AAC TCT TCC TCT TCCTCT TTC TCT TCC CCG ACT TCC GCT GGT 192 Ala Thr Asn Ser Ser Ser Ser SerPhe Ser Ser Pro Thr Ser Ala Gly 50 55 60 CGT CAC GTT CGT TCT TAC AAC CACCTG CAG GGT GAC GTT CGT TGG CGT 240 Arg His Val Arg Ser Tyr Asn His LeuGln Gly Asp Val Arg Trp Arg 65 70 75 80 AAA CTG TTC TCT TTC ACC AAA TACTTC CTG AAA ATC GAA AAA AAC GGT 288 Lys Leu Phe Ser Phe Thr Lys Tyr PheLeu Lys Ile Glu Lys Asn Gly 85 90 95 AAA GTT TCT GGG ACC AAG AAG GAG AACTGC CCG TAC AGC ATC CTG GAG 336 Lys Val Ser Gly Thr Lys Lys Glu Asn CysPro Tyr Ser Ile Leu Glu 100 105 110 ATA ACA TCA GTA GAA ATC GGA GTT GTTGCC GTC AAA GCC ATT AAC AGC 384 Ile Thr Ser Val Glu Ile Gly Val Val AlaVal Lys Ala Ile Asn Ser 115 120 125 AAC TAT TAC TTA GCC ATG AAC AAG AAGGGG AAA CTC TAT GGC TCA AAA 432 Asn Tyr Tyr Leu Ala Met Asn Lys Lys GlyLys Leu Tyr Gly Ser Lys 130 135 140 GAA TTT AAC AAT GAC TGT AAG CTG AAGGAG AGG ATA GAG GAA AAT GGA 480 Glu Phe Asn Asn Asp Cys Lys Leu Lys GluArg Ile Glu Glu Asn Gly 145 150 155 160 TAC AAT ACC TAT GCA TCA TTT AACTGG CAG CAT AAT GGG AGG CAA ATG 528 Tyr Asn Thr Tyr Ala Ser Phe Asn TrpGln His Asn Gly Arg Gln Met 165 170 175 TAT GTG GCA TTG AAT GGA AAA GGAGCT CCA AGG AGA GGA CAG AAA ACA 576 Tyr Val Ala Leu Asn Gly Lys Gly AlaPro Arg Arg Gly Gln Lys Thr 180 185 190 CGA AGG AAA AAC ACC TCT GCT CACTTT CTT CCA ATG GTG GTA CAC TCA 624 Arg Arg Lys Asn Thr Ser Ala His PheLeu Pro Met Val Val His Ser 195 200 205 TAG 627 208 amino acids aminoacid linear protein 39 Met Trp Lys Trp Ile Leu Thr His Cys Ala Ser AlaPhe Pro His Leu 1 5 10 15 Pro Gly Cys Cys Cys Cys Cys Phe Leu Leu LeuPhe Leu Val Ser Ser 20 25 30 Val Pro Val Thr Cys Gln Ala Leu Gly Gln AspMet Val Ser Pro Glu 35 40 45 Ala Thr Asn Ser Ser Ser Ser Ser Phe Ser SerPro Thr Ser Ala Gly 50 55 60 Arg His Val Arg Ser Tyr Asn His Leu Gln GlyAsp Val Arg Trp Arg 65 70 75 80 Lys Leu Phe Ser Phe Thr Lys Tyr Phe LeuLys Ile Glu Lys Asn Gly 85 90 95 Lys Val Ser Gly Thr Lys Lys Glu Asn CysPro Tyr Ser Ile Leu Glu 100 105 110 Ile Thr Ser Val Glu Ile Gly Val ValAla Val Lys Ala Ile Asn Ser 115 120 125 Asn Tyr Tyr Leu Ala Met Asn LysLys Gly Lys Leu Tyr Gly Ser Lys 130 135 140 Glu Phe Asn Asn Asp Cys LysLeu Lys Glu Arg Ile Glu Glu Asn Gly 145 150 155 160 Tyr Asn Thr Tyr AlaSer Phe Asn Trp Gln His Asn Gly Arg Gln Met 165 170 175 Tyr Val Ala LeuAsn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr 180 185 190 Arg Arg LysAsn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 195 200 205 38 basepairs nucleic acid single linear cDNA 40 TTTCATGACT TGTCAAGCTCTGGGTCAAGA TATGGTTC 38 28 base pairs nucleic acid single linear cDNA 41GCCCAAGCTT CCACAAACGT TGCCTTCC 28 525 base pairs nucleic acid doubleboth DNA (genomic) CDS 1..522 42 ATG ACC TGC CAG GCT CTG GGT CAG GAC ATGGTT TCT CCG GAA GCT ACC 48 Met Thr Cys Gln Ala Leu Gly Gln Asp Met ValSer Pro Glu Ala Thr 1 5 10 15 AAC TCT TCC TCT TCC TCT TTC TCT TCC CCGTCT TCC GCT GGT CGT CAC 96 Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro SerSer Ala Gly Arg His 20 25 30 GTT CGT TCT TAC AAC CAC CTG CAG GGT GAC GTTCGT TGG CGT AAA CTG 144 Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val ArgTrp Arg Lys Leu 35 40 45 TTC TCT TTC ACC AAA TAC TTC CTG AAA ATC GAA AAAAAC GGT AAA GTT 192 Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys AsnGly Lys Val 50 55 60 TCT GGG ACC AAG AAG GAG AAC TGC CCG TAC AGC ATC CTGGAG ATA ACA 240 Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu GluIle Thr 65 70 75 80 TCA GTA GAA ATC GGA GTT GTT GCC GTC AAA GCC ATT AACAGC AAC TAT 288 Ser Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn SerAsn Tyr 85 90 95 TAC TTA GCC ATG AAC AAG AAG GGG AAA CTC TAT GGC TCA AAAGAA TTT 336 Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys GluPhe 100 105 110 AAC AAT GAC TGT AAG CTG AAG GAG AGG ATA GAG GAA AAT GGATAC AAT 384 Asn Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly TyrAsn 115 120 125 ACC TAT GCA TCA TTT AAC TGG CAG CAT AAT GGG AGG CAA ATGTAT GTG 432 Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met TyrVal 130 135 140 GCA TTG AAT GGA AAA GGA GCT CCA AGG AGA GGA CAG AAA ACACGA AGG 480 Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr ArgArg 145 150 155 160 AAA AAC ACC TCT GCT CAC TTT CTT CCA ATG GTG GTA CACTCA 522 Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170TAG 525 174 amino acids amino acid linear protein 43 Met Thr Cys Gln AlaLeu Gly Gln Asp Met Val Ser Pro Glu Ala Thr 1 5 10 15 Asn Ser Ser SerSer Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His 20 25 30 Val Arg Ser TyrAsn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 Phe Ser Phe ThrLys Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys Val 50 55 60 Ser Gly Thr LysLys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr 65 70 75 80 Ser Val GluIle Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr 85 90 95 Tyr Leu AlaMet Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 Asn AsnAsp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn 115 120 125 ThrTyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 130 135 140Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg 145 150155 160 Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 17045 base pairs nucleic acid single linear cDNA 44 TCAGTGAATT CATTAAAGAGGAGAAATTAA TCATGACTTG CCAGG 45 48 base pairs nucleic acid single linearcDNA 45 TCATGACTTG CCAGGCACTG GGTCAAGACA TGGTTTCCCC GGAAGCTA 48 48 basepairs nucleic acid single linear cDNA 46 GCTTCAGCAG CCCATCTAGCGCAGGTCGTC ACGTTCGCTC TTACAACC 48 48 base pairs nucleic acid singlelinear cDNA 47 GTTCGTTGGC GCAAACTGTT CAGCTTTACC AAGTACTTCC TGAAAATC 4828 base pairs nucleic acid single linear cDNA 48 TCGAAAAAAA CGGTAAAGTTTCTGGGAC 28 48 base pairs nucleic acid single linear cDNA 49 GATGGGCTGCTGAAGCTAGA GCTGGAGCTG TTGGTAGCTT CCGGGGAA 48 45 base pairs nucleic acidsingle linear cDNA 50 AACAGTTTGC GCCAACGAAC ATCACCCTGT AAGTGGTTGT AAGAG45 47 base pairs nucleic acid single linear cDNA 51 TTCTTGGTCCCAGAAACTTT ACCGTTTTTT TCGATTTTCA GGAAGTA 47 24 base pairs nucleic acidsingle linear cDNA 52 TTCTTGGTCC CAGAAACTTT ACCG 24 45 base pairsnucleic acid single linear cDNA 53 AGATCAGGCT TCTATTATTA TGAGTGTACCACCATTGGAA GAAAG 45 525 base pairs nucleic acid double both DNA(genomic) CDS 1..522 54 ATG ACT TGC CAG GCA CTG GGT CAA GAC ATG GTT TCCCCG GAA GCT ACC 48 Met Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser ProGlu Ala Thr 1 5 10 15 AAC AGC TCC AGC TCT AGC TTC AGC AGC CCA TCT AGCGCA GGT CGT CAC 96 Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser AlaGly Arg His 20 25 30 GTT CGC TCT TAC AAC CAC TTA CAG GGT GAT GTT CGT TGGCGC AAA CTG 144 Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp ArgLys Leu 35 40 45 TTC AGC TTT ACC AAG TAC TTC CTG AAA ATC GAA AAA AAC GGTAAA GTT 192 Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly LysVal 50 55 60 TCT GGG ACC AAG AAG GAG AAC TGC CCG TAC AGC ATC CTG GAG ATAACA 240 Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr65 70 75 80 TCA GTA GAA ATC GGA GTT GTT GCC GTC AAA GCC ATT AAC AGC AACTAT 288 Ser Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr85 90 95 TAC TTA GCC ATG AAC AAG AAG GGG AAA CTC TAT GGC TCA AAA GAA TTT336 Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100105 110 AAC AAT GAC TGT AAG CTG AAG GAG AGG ATA GAG GAA AAT GGA TAC AAT384 Asn Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn 115120 125 ACC TAT GCA TCA TTT AAC TGG CAG CAT AAT GGG AGG CAA ATG TAT GTG432 Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 130135 140 GCA TTG AAT GGA AAA GGA GCT CCA AGG AGA GGA CAG AAA ACA CGA AGG480 Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg 145150 155 160 AAA AAC ACC TCT GCT CAC TTT CTT CCA ATG GTG GTA CAC TCA 522Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170 TAG 525174 amino acids amino acid linear protein 55 Met Thr Cys Gln Ala Leu GlyGln Asp Met Val Ser Pro Glu Ala Thr 1 5 10 15 Asn Ser Ser Ser Ser SerPhe Ser Ser Pro Ser Ser Ala Gly Arg His 20 25 30 Val Arg Ser Tyr Asn HisLeu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 Phe Ser Phe Thr Lys TyrPhe Leu Lys Ile Glu Lys Asn Gly Lys Val 50 55 60 Ser Gly Thr Lys Lys GluAsn Cys Pro Tyr Ser Ile Leu Glu Ile Thr 65 70 75 80 Ser Val Glu Ile GlyVal Val Ala Val Lys Ala Ile Asn Ser Asn Tyr 85 90 95 Tyr Leu Ala Met AsnLys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 Asn Asn Asp CysLys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn 115 120 125 Thr Tyr AlaSer Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 130 135 140 Ala LeuAsn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg 145 150 155 160Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170 35 basepairs nucleic acid single linear cDNA 56 GGACCCTCAT GACCTGCCAGGCTCTGGGTC AGGAC 35 28 base pairs nucleic acid single linear cDNA 57GGACAGCCAT GGCTGGTCGT CACGTTCG 28 29 base pairs nucleic acid singlelinear cDNA 58 GGACAGCCAT GGTTCGTTGG CGTAAACTG 29 31 base pairs nucleicacid single linear cDNA 59 GGACAGCCAT GGAAAAAAAC GGTAAAGTTT C 31 29 basepairs nucleic acid single linear cDNA 60 GGACCCCCAT GGAGAACTGC CCGTAGAGC29 32 base pairs nucleic acid single linear cDNA 61 GGACCCCCATGGTCAAAGCC ATTAACAGCA AC 32 33 base pairs nucleic acid single linearcDNA 62 GGACCCCCAT GGGGAAACTC TATGGCTCAA AAG 33 37 base pairs nucleicacid single linear cDNA 63 CTGCCCAAGC TTATTATGAG TGTACCACCA TTGGAAG 3736 base pairs nucleic acid single linear cDNA 64 CTGCCCAAGC TTATTACTTCAGCTTACAGT CATTGT 36 525 base pairs nucleic acid double both DNA(genomic) CDS 1..522 65 ATG ACC TGC CAG GCT CTG GGT CAG GAC ATG GTT TCTCCG GAA GCT ACC 48 Met Thr Cys Gln Ala Leu Gly Gln Asp Met Val Ser ProGlu Ala Thr 1 5 10 15 AAC TCT TCC TCT TCC TCT TTC TCT TCC CCG TCT TCCGCT GGT CGT CAC 96 Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro Ser Ser AlaGly Arg His 20 25 30 GTT CGT TCT TAC AAC CAC CTG CAG GGT GAC GTT CGT TGGCGT AAA CTG 144 Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp ArgLys Leu 35 40 45 TTC TCT TTC ACC AAA TAC TTC CTG AAA ATC GAA AAA AAC GGTAAA GTT 192 Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly LysVal 50 55 60 TCT GGG ACC AAG AAG GAG AAC TGC CCG TAC AGC ATC CTG GAG ATAACA 240 Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr65 70 75 80 TCA GTA GAA ATC GGA GTT GTT GCC GTC AAA GCC ATT AAC AGC AACTAT 288 Ser Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr85 90 95 TAC TTA GCC ATG AAC AAG AAG GGG AAA CTC TAT GGC TCA AAA GAA TTT336 Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100105 110 AAC AAT GAC TGT AAG CTG AAG GAG AGG ATA GAG GAA AAT GGA TAC AAT384 Asn Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn 115120 125 ACC TAT GCA TCA TTT AAC TGG CAG CAT AAT GGG AGG CAA ATG TAT GTG432 Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 130135 140 GCA TTG AAT GGA AAA GGA GCT CCA AGG AGA GGA CAG AAA ACA CGA AGG480 Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg 145150 155 160 AAA AAC ACC TCT GCT CAC TTT CTT CCA ATG GTG GTA CAC TCA 522Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170 TAG 525174 amino acids amino acid linear protein 66 Met Thr Cys Gln Ala Leu GlyGln Asp Met Val Ser Pro Glu Ala Thr 1 5 10 15 Asn Ser Ser Ser Ser SerPhe Ser Ser Pro Ser Ser Ala Gly Arg His 20 25 30 Val Arg Ser Tyr Asn HisLeu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 Phe Ser Phe Thr Lys TyrPhe Leu Lys Ile Glu Lys Asn Gly Lys Val 50 55 60 Ser Gly Thr Lys Lys GluAsn Cys Pro Tyr Ser Ile Leu Glu Ile Thr 65 70 75 80 Ser Val Glu Ile GlyVal Val Ala Val Lys Ala Ile Asn Ser Asn Tyr 85 90 95 Tyr Leu Ala Met AsnLys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 Asn Asn Asp CysLys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn 115 120 125 Thr Tyr AlaSer Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val 130 135 140 Ala LeuAsn Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg 145 150 155 160Lys Asn Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 165 170 444 basepairs nucleic acid double both DNA (genomic) CDS 1..441 67 ATG GCT GGTCGT CAC GTT CGT TCT TAC AAC CAC CTG CAG GGT GAC GTT 48 Met Ala Gly ArgHis Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val 1 5 10 15 CGT TGG CGTAAA CTG TTC TCT TTC ACC AAA TAC TTC CTG AAA ATC GAA 96 Arg Trp Arg LysLeu Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu 20 25 30 AAA AAC GGT AAAGTT TCT GGG ACC AAG AAG GAG AAC TGC CCG TAC AGC 144 Lys Asn Gly Lys ValSer Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser 35 40 45 ATC CTG GAG ATA ACATCA GTA GAA ATC GGA GTT GTT GCC GTC AAA GCC 192 Ile Leu Glu Ile Thr SerVal Glu Ile Gly Val Val Ala Val Lys Ala 50 55 60 ATT AAC AGC AAC TAT TACTTA GCC ATG AAC AAG AAG GGG AAA CTC TAT 240 Ile Asn Ser Asn Tyr Tyr LeuAla Met Asn Lys Lys Gly Lys Leu Tyr 65 70 75 80 GGC TCA AAA GAA TTT AACAAT GAC TGT AAG CTG AAG GAG AGG ATA GAG 288 Gly Ser Lys Glu Phe Asn AsnAsp Cys Lys Leu Lys Glu Arg Ile Glu 85 90 95 GAA AAT GGA TAC AAT ACC TATGCA TCA TTT AAC TGG CAG CAT AAT GGG 336 Glu Asn Gly Tyr Asn Thr Tyr AlaSer Phe Asn Trp Gln His Asn Gly 100 105 110 AGG CAA ATG TAT GTG GCA TTGAAT GGA AAA GGA GCT CCA AGG AGA GGA 384 Arg Gln Met Tyr Val Ala Leu AsnGly Lys Gly Ala Pro Arg Arg Gly 115 120 125 CAG AAA ACA CGA AGG AAA AACACC TCT GCT CAC TTT CTT CCA ATG GTG 432 Gln Lys Thr Arg Arg Lys Asn ThrSer Ala His Phe Leu Pro Met Val 130 135 140 GTA CAC TCA TAG 444 Val HisSer 145 147 amino acids amino acid linear protein 68 Met Ala Gly Arg HisVal Arg Ser Tyr Asn His Leu Gln Gly Asp Val 1 5 10 15 Arg Trp Arg LysLeu Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu 20 25 30 Lys Asn Gly LysVal Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser 35 40 45 Ile Leu Glu IleThr Ser Val Glu Ile Gly Val Val Ala Val Lys Ala 50 55 60 Ile Asn Ser AsnTyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr 65 70 75 80 Gly Ser LysGlu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu 85 90 95 Glu Asn GlyTyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His Asn Gly 100 105 110 Arg GlnMet Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly 115 120 125 GlnLys Thr Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro Met Val 130 135 140Val His Ser 145 402 base pairs nucleic acid double both DNA (genomic)CDS 1..399 69 ATG GTT CGT TGG CGT AAA CTG TTC TCT TTC ACC AAA TAC TTCCTG AAA 48 Met Val Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr Phe LeuLys 1 5 10 15 ATC GAA AAA AAC GGT AAA GTT TCT GGG ACC AAG AAG GAG AACTGC CCG 96 Ile Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn CysPro 20 25 30 TAC AGC ATC CTG GAG ATA ACA TCA GTA GAA ATC GGA GTT GTT GCCGTC 144 Tyr Ser Ile Leu Glu Ile Thr Ser Val Glu Ile Gly Val Val Ala Val35 40 45 AAA GCC ATT AAC AGC AAC TAT TAC TTA GCC ATG AAC AAG AAG GGG AAA192 Lys Ala Ile Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys 5055 60 CTC TAT GGC TCA AAA GAA TTT AAC AAT GAC TGT AAG CTG AAG GAG AGG240 Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg 6570 75 80 ATA GAG GAA AAT GGA TAC AAT ACC TAT GCA TCA TTT AAC TGG CAG CAT288 Ile Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His 8590 95 AAT GGG AGG CAA ATG TAT GTG GCA TTG AAT GGA AAA GGA GCT CCA AGG336 Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg 100105 110 AGA GGA CAG AAA ACA CGA AGG AAA AAC ACC TCT GCT CAC TTT CTT CCA384 Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Ala His Phe Leu Pro 115120 125 ATG GTG GTA CAC TCA TAG 402 Met Val Val His Ser 130 133 aminoacids amino acid linear protein 70 Met Val Arg Trp Arg Lys Leu Phe SerPhe Thr Lys Tyr Phe Leu Lys 1 5 10 15 Ile Glu Lys Asn Gly Lys Val SerGly Thr Lys Lys Glu Asn Cys Pro 20 25 30 Tyr Ser Ile Leu Glu Ile Thr SerVal Glu Ile Gly Val Val Ala Val 35 40 45 Lys Ala Ile Asn Ser Asn Tyr TyrLeu Ala Met Asn Lys Lys Gly Lys 50 55 60 Leu Tyr Gly Ser Lys Glu Phe AsnAsn Asp Cys Lys Leu Lys Glu Arg 65 70 75 80 Ile Glu Glu Asn Gly Tyr AsnThr Tyr Ala Ser Phe Asn Trp Gln His 85 90 95 Asn Gly Arg Gln Met Tyr ValAla Leu Asn Gly Lys Gly Ala Pro Arg 100 105 110 Arg Gly Gln Lys Thr ArgArg Lys Asn Thr Ser Ala His Phe Leu Pro 115 120 125 Met Val Val His Ser130 354 base pairs nucleic acid double both DNA (genomic) CDS 1..351 71ATG GAA AAA AAC GGT AAA GTT TCT GGG ACC AAG AAG GAG AAC TGC CCG 48 MetGlu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys Pro 1 5 10 15TAC AGC ATC CTG GAG ATA ACA TCA GTA GAA ATC GGA GTT GTT GCC GTC 96 TyrSer Ile Leu Glu Ile Thr Ser Val Glu Ile Gly Val Val Ala Val 20 25 30 AAAGCC ATT AAC AGC AAC TAT TAC TTA GCC ATG AAC AAG AAG GGG AAA 144 Lys AlaIle Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys 35 40 45 CTC TATGGC TCA AAA GAA TTT AAC AAT GAC TGT AAG CTG AAG GAG AGG 192 Leu Tyr GlySer Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys Glu Arg 50 55 60 ATA GAG GAAAAT GGA TAC AAT ACC TAT GCA TCA TTT AAC TGG CAG CAT 240 Ile Glu Glu AsnGly Tyr Asn Thr Tyr Ala Ser Phe Asn Trp Gln His 65 70 75 80 AAT GGG AGGCAA ATG TAT GTG GCA TTG AAT GGA AAA GGA GCT CCA AGG 288 Asn Gly Arg GlnMet Tyr Val Ala Leu Asn Gly Lys Gly Ala Pro Arg 85 90 95 AGA GGA CAG AAAACA CGA AGG AAA AAC ACC TCT GCT CAC TTT CTT CCA 336 Arg Gly Gln Lys ThrArg Arg Lys Asn Thr Ser Ala His Phe Leu Pro 100 105 110 ATG GTG GTA CACTCA TAG 354 Met Val Val His Ser 115 117 amino acids amino acid linearprotein 72 Met Glu Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn CysPro 1 5 10 15 Tyr Ser Ile Leu Glu Ile Thr Ser Val Glu Ile Gly Val ValAla Val 20 25 30 Lys Ala Ile Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys LysGly Lys 35 40 45 Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu LysGlu Arg 50 55 60 Ile Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn TrpGln His 65 70 75 80 Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly Lys GlyAla Pro Arg 85 90 95 Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Ala HisPhe Leu Pro 100 105 110 Met Val Val His Ser 115 321 base pairs nucleicacid double linear DNA (genomic) CDS 1..318 73 ATG GAG AAC TGC CCG TACAGC ATC CTG GAG ATA ACA TCA GTA GAA ATC 48 Met Glu Asn Cys Pro Tyr SerIle Leu Glu Ile Thr Ser Val Glu Ile 185 190 195 200 GGA GTT GTT GCC GTCAAA GCC ATT AAC AGC AAC TAT TAC TTA GCC ATG 96 Gly Val Val Ala Val LysAla Ile Asn Ser Asn Tyr Tyr Leu Ala Met 205 210 215 AAC AAG AAG GGG AAACTC TAT GGC TCA AAA GAA TTT AAC AAT GAC TGT 144 Asn Lys Lys Gly Lys LeuTyr Gly Ser Lys Glu Phe Asn Asn Asp Cys 220 225 230 AAG CTG AAG GAG AGGATA GAG GAA AAT GGA TAC AAT ACC TAT GCA TCA 192 Lys Leu Lys Glu Arg IleGlu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser 235 240 245 TTT AAC TGG CAG CATAAT GGG AGG CAA ATG TAT GTG GCA TTG AAT GGA 240 Phe Asn Trp Gln His AsnGly Arg Gln Met Tyr Val Ala Leu Asn Gly 250 255 260 AAA GGA GCT CCA AGGAGA GGA CAG AAA ACA CGA AGG AAA AAC ACC TCT 288 Lys Gly Ala Pro Arg ArgGly Gln Lys Thr Arg Arg Lys Asn Thr Ser 265 270 275 280 GCT CAC TTT CTTCCA ATG GTG GTA CAC TCA TAG 321 Ala His Phe Leu Pro Met Val Val His Ser285 290 106 amino acids amino acid linear protein 74 Met Glu Asn Cys ProTyr Ser Ile Leu Glu Ile Thr Ser Val Glu Ile 1 5 10 15 Gly Val Val AlaVal Lys Ala Ile Asn Ser Asn Tyr Tyr Leu Ala Met 20 25 30 Asn Lys Lys GlyLys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys 35 40 45 Lys Leu Lys GluArg Ile Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser 50 55 60 Phe Asn Trp GlnHis Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly 65 70 75 80 Lys Gly AlaPro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser 85 90 95 Ala His PheLeu Pro Met Val Val His Ser 100 105 264 base pairs nucleic acid doubleboth DNA (genomic) CDS 1..261 75 ATG GTC AAA GCC ATT AAC AGC AAC TAT TACTTA GCC ATG AAC AAG AAG 48 Met Val Lys Ala Ile Asn Ser Asn Tyr Tyr LeuAla Met Asn Lys Lys 1 5 10 15 GGG AAA CTC TAT GGC TCA AAA GAA TTT AACAAT GAC TGT AAG CTG AAG 96 Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn AsnAsp Cys Lys Leu Lys 20 25 30 GAG AGG ATA GAG GAA AAT GGA TAC AAT ACC TATGCA TCA TTT AAC TGG 144 Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr Tyr AlaSer Phe Asn Trp 35 40 45 CAG CAT AAT GGG AGG CAA ATG TAT GTG GCA TTG AATGGA AAA GGA GCT 192 Gln His Asn Gly Arg Gln Met Tyr Val Ala Leu Asn GlyLys Gly Ala 50 55 60 CCA AGG AGA GGA CAG AAA ACA CGA AGG AAA AAC ACC TCTGCT CAC TTT 240 Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser AlaHis Phe 65 70 75 80 CTT CCA ATG GTG GTA CAC TCA TAG 264 Leu Pro Met ValVal His Ser 85 87 amino acids amino acid linear protein 76 Met Val LysAla Ile Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys 1 5 10 15 Gly LysLeu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu Lys 20 25 30 Glu ArgIle Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn Trp 35 40 45 Gln HisAsn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly Lys Gly Ala 50 55 60 Pro ArgArg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Ala His Phe 65 70 75 80 LeuPro Met Val Val His Ser 85 219 base pairs nucleic acid double both DNA(genomic) CDS 1..216 77 ATG GGG AAA CTC TAT GGC TCA AAA GAA TTT AAC AATGAC TGT AAG CTG 48 Met Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn AspCys Lys Leu 1 5 10 15 AAG GAG AGG ATA GAG GAA AAT GGA TAC AAT ACC TATGCA TCA TTT AAC 96 Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr Tyr AlaSer Phe Asn 20 25 30 TGG CAG CAT AAT GGG AGG CAA ATG TAT GTG GCA TTG AATGGA AAA GGA 144 Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala Leu Asn GlyLys Gly 35 40 45 GCT CCA AGG AGA GGA CAG AAA ACA CGA AGG AAA AAC ACC TCTGCT CAC 192 Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser AlaHis 50 55 60 TTT CTT CCA ATG GTG GTA CAC TCA TAG 219 Phe Leu Pro Met ValVal His Ser 65 70 72 amino acids amino acid linear protein 78 Met GlyLys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys Leu 1 5 10 15 LysGlu Arg Ile Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser Phe Asn 20 25 30 TrpGln His Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly Lys Gly 35 40 45 AlaPro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser Ala His 50 55 60 PheLeu Pro Met Val Val His Ser 65 70 357 base pairs nucleic acid doubleboth DNA (genomic) CDS 1..357 79 ATG ACC TGC CAG GCT CTG GGT CAG GAC ATGGTT TCT CCG GAA GCT ACC 48 Met Thr Cys Gln Ala Leu Gly Gln Asp Met ValSer Pro Glu Ala Thr 1 5 10 15 AAC TCT TCC TCT TCC TCT TTC TCT TCC CCGTCT TCC GCT GGT CGT CAC 96 Asn Ser Ser Ser Ser Ser Phe Ser Ser Pro SerSer Ala Gly Arg His 20 25 30 GTT CGT TCT TAC AAC CAC CTG CAG GGT GAC GTTCGT TGG CGT AAA CTG 144 Val Arg Ser Tyr Asn His Leu Gln Gly Asp Val ArgTrp Arg Lys Leu 35 40 45 TTC TCT TTC ACC AAA TAC TTC CTG AAA ATC GAA AAAAAC GGT AAA GTT 192 Phe Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys AsnGly Lys Val 50 55 60 TCT GGG ACC AAG AAG GAG AAC TGC CCG TAC AGC ATC CTGGAG ATA ACA 240 Ser Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu GluIle Thr 65 70 75 80 TCA GTA GAA ATC GGA GTT GTT GCC GTC AAA GCC ATT AACAGC AAC TAT 288 Ser Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn SerAsn Tyr 85 90 95 TAC TTA GCC ATG AAC AAG AAG GGG AAA CTC TAT GGC TCA AAAGAA TTT 336 Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys GluPhe 100 105 110 AAC AAT GAC TGT AAG CTG AAG 357 Asn Asn Asp Cys Lys LeuLys 115 119 amino acids amino acid linear protein 80 Met Thr Cys Gln AlaLeu Gly Gln Asp Met Val Ser Pro Glu Ala Thr 1 5 10 15 Asn Ser Ser SerSer Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His 20 25 30 Val Arg Ser TyrAsn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu 35 40 45 Phe Ser Phe ThrLys Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys Val 50 55 60 Ser Gly Thr LysLys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr 65 70 75 80 Ser Val GluIle Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr 85 90 95 Tyr Leu AlaMet Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe 100 105 110 Asn AsnAsp Cys Lys Leu Lys 115 276 base pairs nucleic acid double both DNA(genomic) CDS 1..276 81 ATG GCT GGT CGT CAC GTT CGT TCT TAC AAC CAC CTGCAG GGT GAC GTT 48 Met Ala Gly Arg His Val Arg Ser Tyr Asn His Leu GlnGly Asp Val 1 5 10 15 CGT TGG CGT AAA CTG TTC TCT TTC ACC AAA TAC TTCCTG AAA ATC GAA 96 Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr Phe LeuLys Ile Glu 20 25 30 AAA AAC GGT AAA GTT TCT GGG ACC AAG AAG GAG AAC TGCCCG TAC AGC 144 Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu Asn Cys ProTyr Ser 35 40 45 ATC CTG GAG ATA ACA TCA GTA GAA ATC GGA GTT GTT GCC GTCAAA GCC 192 Ile Leu Glu Ile Thr Ser Val Glu Ile Gly Val Val Ala Val LysAla 50 55 60 ATT AAC AGC AAC TAT TAC TTA GCC ATG AAC AAG AAG GGG AAA CTCTAT 240 Ile Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr65 70 75 80 GGC TCA AAA GAA TTT AAC AAT GAC TGT AAG CTG AAG 276 Gly SerLys Glu Phe Asn Asn Asp Cys Lys Leu Lys 85 90 92 amino acids amino acidlinear protein 82 Met Ala Gly Arg His Val Arg Ser Tyr Asn His Leu GlnGly Asp Val 1 5 10 15 Arg Trp Arg Lys Leu Phe Ser Phe Thr Lys Tyr PheLeu Lys Ile Glu 20 25 30 Lys Asn Gly Lys Val Ser Gly Thr Lys Lys Glu AsnCys Pro Tyr Ser 35 40 45 Ile Leu Glu Ile Thr Ser Val Glu Ile Gly Val ValAla Val Lys Ala 50 55 60 Ile Asn Ser Asn Tyr Tyr Leu Ala Met Asn Lys LysGly Lys Leu Tyr 65 70 75 80 Gly Ser Lys Glu Phe Asn Asn Asp Cys Lys LeuLys 85 90 525 base pairs nucleic acid double both DNA (genomic) 83ATGACCTCTC AGGCTCTGGG TCAGGACATG GTTTCTCCGG AAGCTACCAA CTCTTCCTCT 60TCCTCTTTCT CTTCCCCGTC TTCCGCTGGT CGTCACGTTC GTTCTTACAA CCACCTGCAG 120GGTGACGTTC GTTGGCGTAA ACTGTTCTCT TTCACCAAAT ACTTCCTGAA AATCGAAAAA 180AACGGTAAAG TTTCTGGGAC CAAGAAGGAG AACTCTCCGT ACAGCATCCT GGAGATAACA 240TCAGTAGAAA TCGGAGTTGT TGCCGTCAAA GCCATTAACA GCAACTATTA CTTAGCCATG 300AACAAGAAGG GGAAACTCTA TGGCTCAAAA GAATTTAACA ATGACTGTAA GCTGAAGGAG 360AGGATAGAGG AAAATGGATA CAATACCTAT GCATCATTTA ACTGGCAGCA TAATGGGAGG 420CAAATGTATG TGGCATTGAA TGGAAAAGGA GCTCCAAGGA GAGGACAGAA AACACGAAGG 480AAAAACACCT CTGCTCACTT TCTTCCAATG GTGGTACACT CATAG 525 525 base pairsnucleic acid double both DNA (genomic) 84 ATGACCTGCC AGGCTCTGGGTCAGGACATG GTTTCTCCGG AAGCTACCAA CTCTTCCTCT 60 TCCTCTTTCT CTTCCCCGTCTTCCGCTGGT CGTCACGTTC GTTCTTACAA CCACCTGCAG 120 GGTGACGTTC GTTGGCGTAAACTGTTCTCT TTCACCAAAT ACTTCCTGAA AATCGAAAAA 180 AACGGTAAAG TTTCTGGGACCAAGAAGGAG AACTCTCCGT ACAGCATCCT GGAGATAACA 240 TCAGTAGAAA TCGGAGTTGTTGCCGTCAAA GCCATTAACA GCAACTATTA CTTAGCCATG 300 AACAAGAAGG GGAAACTCTATGGCTCAAAA GAATTTAACA ATGACTGTAA GCTGAAGGAG 360 AGGATAGAGG AAAATGGATACAATACCTAT GCATCATTTA ACTGGCAGCA TAATGGGAGG 420 CAAATGTATG TGGCATTGAATGGAAAAGGA GCTCCAAGGA GAGGACAGAA AACACGAAGG 480 AAAAACACCT CTGCTCACTTTCTTCCAATG GTGGTACACT CATAG 525 29 base pairs nucleic acid single linearcDNA 85 GGACCCTCAT GACCTCTCAG GCTCTGGGT 29 21 base pairs nucleic acidsingle linear cDNA 86 AAGGAGAACT CTCCGTACAG C 21 21 base pairs nucleicacid single linear cDNA 87 GCTGTACGGT CTGTTCTCCT T 21 35 base pairsnucleic acid single linear cDNA 88 GGACCCTCAT GACCTGCCAG GCTCTGGGTCAGGAC 35 37 base pairs nucleic acid single linear cDNA 89 CTGCCCAAGCTTATTATGAG TGTACCACCA TTGGAAG 37 33 base pairs nucleic acid singlelinear cDNA 90 AAAGGATCCT GCCAGGCTCT GGGTCAGGAC ATG 33 32 base pairsnucleic acid single linear cDNA 91 GCGGCACATG TCTTACAACC ACCTGCAGGG TG32 28 base pairs nucleic acid single linear cDNA 92 GGGCCCAAGCTTATGAGTGT ACCACCAT 28 36 base pairs nucleic acid single linear cDNA 93CCGGCGGATC CCATATGTCT TACAACCACC TGCAGG 36 35 base pairs nucleic acidsingle linear cDNA 94 CCGGCGGTAC CTTATTATGA GTGTACCACC ATTGG 35 426 basepairs nucleic acid double linear cDNA 95 ATGTCTTACA ACCACCTGCAGGGTGACGTT CGTTGGCGTA AACTGTTCTC TTTCACCAAA 60 TACTTCCTGA AAATCGAAAAAAACGGTAAA GTTTCTGGGA CCAAGAAGGA GAACTGCCCG 120 TACAGCATCC TGGAGATAACATCAGTAGAA ATCGGAGTTG TTGCCGTCAA AGCCATTAAC 180 AGCAACTATT ACTTAGCCATGAACAAGAAG GGGAAACTCT ATGGCTCAAA AGAATTTAAC 240 AATGACTGTA AGCTGAAGGAGAGGATAGAG GAAAATGGAT ACAATACCTA TGCATCATTT 300 AACTGGCAGC ATAATGGGAGGCAAATGTAT GTGGCATTGA ATGGAAAAGG AGCTCCAAGG 360 AGAGGACAGA AAACACGAAGGAAAAACACC TCTGCTCACT TTCTTCCAAT GGTGGTACAC 420 TCATAA 426 141 aminoacids amino acid single Not Relevant protein 96 Met Ser Tyr Asn His LeuGln Gly Asp Val Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys TyrPhe Leu Lys Ile Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys GluAsn Cys Pro Tyr Ser Ile Leu Glu Ile Thr Ser 35 40 45 Val Glu Ile Gly ValVal Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn LysLys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys LysLeu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Ala Ser PheAsn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala 100 105 110 Leu Asn GlyLys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys 115 120 125 Asn ThrSer Ala His Phe Leu Pro Met Val Val His Ser 130 135 140 20 base pairsnucleic acid single linear cDNA 97 CAACCACCTG CAGGGTGACG 20 78 basepairs nucleic acid single linear cDNA 98 AACGGTCGAC AAATGTATGTGGCACTGAAC GGTAAAGGTG CTCCACGTCG TGGTCAGAAA 60 ACCCGTCGTA AAAACACC 78 76base pairs nucleic acid single linear cDNA 99 GGGCCCAAGC TTAAGAGTGTACCACCATTG GCAGAAAGTG AGCAGAGGTG TTTTTACGAC 60 GGGTTTTCTG ACCACG 76 23base pairs nucleic acid single linear cDNA 100 GCCACATACA TTTGTCGACC GTT23 19 base pairs nucleic acid single linear cDNA 101 GGGCCCAAGCTTAAGAGTG 19 23 base pairs nucleic acid single linear cDNA 102GCCACATACA TTTGTCGACC GTT 23 90 base pairs nucleic acid single linearcDNA 103 CTGCAGGGTG ACGTTCGTTG GCGTAAACTG TTCTCCTTCA CCAAATACTTCCTGAAAATC 60 GAAAAAAACG GTAAAGTTTC TGGTACCAAG 90 90 base pairs nucleicacid single linear cDNA 104 AGCTTTAACA GCAACAACAC CGATTTCAAC GGAGGTGATTTCCAGGATGG AGTACGGGCA 60 GTTTTCTTTC TTGGTACCAG AAACTTTACC 90 90 basepairs nucleic acid single linear cDNA 105 GGTGTTGTTG CTGTTAAAGCTATCAACTCC AACTACTACC TGGCTATGAA CAAGAAAGGT 60 AAACTGTACG GTTCCAAAGAATTTAACAAC 90 100 base pairs nucleic acid single linear cDNA 106GTCGACCGTT GTGCTGCCAG TTGAAGGAAG CGTAGGTGTT GTAACCGTTT TCTTCGATAC 60GTTCTTTCAG TTTACAGTCG TTGTTAAATT CTTTGGAACC 100 25 base pairs nucleicacid single linear cDNA 107 GCGGCGTCGA CCGTTGTGCT GCCAG 25 26 base pairsnucleic acid single linear cDNA 108 GCGGCCTGCA GGGTGACGTT CGTTGG 26 36base pairs nucleic acid single linear cDNA 109 CCGGCGGATC CCATATGTCTTACAACCACC TGCAGG 36 34 base pairs nucleic acid single linear cDNA 110CGCGCGATAT CTTATTAAGA GTGTACCACC ATTG 34 426 base pairs nucleic aciddouble linear cDNA 111 ATGTCTTACA ACCACCTGCA GGGTGACGTT CGTTGGCGTAAACTGTTCTC CTTCACCAAA 60 TACTTCCTGA AAATCGAAAA AAACGGTAAA GTTTCTGGTACCAAGAAAGA AAACTGCCCG 120 TACTCCATCC TGGAAATCAC CTCCGTTGAA ATCGGTGTTGTTGCTGTTAA AGCTATCAAC 180 TCCAACTACT ACCTGGCTAT GAACAAGAAA GGTAAACTGTACGGTTCCAA AGAATTTAAC 240 AACGACTGTA AACTGAAAGA ACGTATCGAA GAAAACGGTTACAACACCTA CGCTTCCTTC 300 AACTGGCAGC ACAACGGTCG ACAAATGTAT GTGGCACTGAACGGTAAAGG TGCTCCACGT 360 CGTGGTCAGA AAACCCGTCG TAAAAACACC TCTGCTCACTTTCTGCCAAT GGTGGTACAC 420 TCTTAA 426 141 amino acids amino acid singleNot Relevant protein 112 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg TrpArg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu LysAsn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser IleLeu Glu Ile Thr Ser 35 40 45 Val Glu Ile Gly Val Val Ala Val Lys Ala IleAsn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr GlySer Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg Ile GluGlu Asn Gly Tyr Asn Thr 85 90 95 Tyr Ala Ser Phe Asn Trp Gln His Asn GlyArg Gln Met Tyr Val Ala 100 105 110 Leu Asn Gly Lys Gly Ala Pro Arg ArgGly Gln Lys Thr Arg Arg Lys 115 120 125 Asn Thr Ser Ala His Phe Leu ProMet Val Val His Ser 130 135 140 28 base pairs nucleic acid single linearcDNA 113 CGCGGCCATG GCTCTGGGTC AGGACATG 28 28 base pairs nucleic acidsingle linear cDNA 114 GGGCCCAAGC TTATGAGTGT ACCACCAT 28 516 base pairsnucleic acid double linear cDNA 115 ATGGCTCTGG GTCAAGATAT GGTTTCTCCGGAAGCTACCA ACTCTTCCTC TTCCTCTTTC 60 TCTTCCCCGT CTTCCGCTGG TCGTCACGTTCGTTCTTACA ACCACCTGCA GGGTGACGTT 120 CGTTGGCGTA AACTGTTCTC TTTCACCAAATACTTCCTGA AAATCGAAAA AAACGGTAAA 180 GTTTCTGGGA CCAAGAAGGA GAACTGCCCGTACAGCATCC TGGAGATAAC ATCAGTAGAA 240 ATCGGAGTTG TTGCCGTCAA AGCCATTAACAGCAACTATT ACTTAGCCAT GAACAAGAAG 300 GGGAAACTCT ATGGCTCAAA AGAATTTAACAATGACTGTA AGCTGAAGGA GAGGATAGAG 360 GAAAATGGAT ACAATACCTA TGCATCATTTAACTGGCAGC ATAATGGGAG GCAAATGTAT 420 GTGGCATTGA ATGGAAAAGG AGCTCCAAGGAGAGGACAGA AAACACGAAG GAAAAACACC 480 TCTGCTCACT TTCTTCCAAT GGTGGTACACTCATAA 516 171 amino acids amino acid single Not Relevant protein 116Met Ala Leu Gly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser 1 5 1015 Ser Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser 20 2530 Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe Ser Phe 35 4045 Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys Val Ser Gly Thr 50 5560 Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr Ser Val Glu 65 7075 80 Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr Leu Ala 8590 95 Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp100 105 110 Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr TyrAla 115 120 125 Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val AlaLeu Asn 130 135 140 Gly Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg ArgLys Asn Thr 145 150 155 160 Ser Ala His Phe Leu Pro Met Val Val His Ser165 170 32 base pairs nucleic acid single linear cDNA 117 GCGGCACATGTCTTACAACC ACCTGCAGGG TG 32 75 base pairs nucleic acid single linearcDNA 118 CTGCCCAAGC TTTTATGAGT GTACCACCAT TGGAAGAAAG TGAGCAGAGGTGTTTTTTTC 60 TCGTGTTTTC TGTCC 75 426 base pairs nucleic acid doublelinear cDNA 119 ATGTCTTACA ACCACCTGCA GGGTGACGTT CGTTGGCGTA AACTGTTCTCTTTCACCAAA 60 TACTTCCTGA AAATCGAAAA AAACGGTAAA GTTTCTGGGA CCAAGAAGGAGAACTGCCCG 120 TACAGCATCC TGGAGATAAC ATCAGTAGAA ATCGGAGTTG TTGCCGTCAAAGCCATTAAC 180 AGCAACTATT ACTTAGCCAT GAACAAGAAG GGGAAACTCT ATGGCTCAAAAGAATTTAAC 240 AATGACTGTA AGCTGAAGGA GAGGATAGAG GAAAATGGAT ACAATACCTATGCATCATTT 300 AACTGGCAGC ATAATGGGAG GCAAATGTAT GTGGCATTGA ATGGAAAAGGAGCTCCAAGG 360 AGAGGACAGA AAACACGAGA AAAAAACACC TCTGCTCACT TTCTTCCAATGGTGGTACAC 420 TCATAG 426 141 amino acids amino acid single Not Relevantprotein 120 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys LeuPhe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly LysVal Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu IleThr Ser 35 40 45 Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser AsnTyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys GluPhe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn GlyTyr Asn Thr 85 90 95 Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln MetTyr Val Ala 100 105 110 Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln LysThr Arg Glu Lys 115 120 125 Asn Thr Ser Ala His Phe Leu Pro Met Val ValHis Ser 130 135 140 32 base pairs nucleic acid single linear cDNA 121GCGGCACATG TCTTACAACC ACCTGCAGGG TG 32 75 base pairs nucleic acid singlelinear cDNA 122 CTGCCCAAGC TTTTATGAGT GTACCACCAT TGGAAGAAAG TGAGCAGAGGTGTTTTTCTG 60 TCGTGTTTTC TGTCC 75 426 base pairs nucleic acid doublelinear cDNA 123 ATGTCTTACA ACCACCTGCA GGGTGACGTT CGTTGGCGTA AACTGTTCTCTTTCACCAAA 60 TACTTCCTGA AAATCGAAAA AAACGGTAAA GTTTCTGGGA CCAAGAAGGAGAACTGCCCG 120 TACAGCATCC TGGAGATAAC ATCAGTAGAA ATCGGAGTTG TTGCCGTCAAAGCCATTAAC 180 AGCAACTATT ACTTAGCCAT GAACAAGAAG GGGAAACTCT ATGGCTCAAAAGAATTTAAC 240 AATGACTGTA AGCTGAAGGA GAGGATAGAG GAAAATGGAT ACAATACCTATGCATCATTT 300 AACTGGCAGC ATAATGGGAG GCAAATGTAT GTGGCATTGA ATGGAAAAGGAGCTCCAAGG 360 AGAGGACAGA AAACACGACA GAAAAACACC TCTGCTCACT TTCTTCCAATGGTGGTACAC 420 TCATAG 426 141 amino acids amino acid single Not Relevantprotein 124 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys LeuPhe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly LysVal Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu IleThr Ser 35 40 45 Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser AsnTyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys GluPhe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn GlyTyr Asn Thr 85 90 95 Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln MetTyr Val Ala 100 105 110 Leu Asn Gly Lys Gly Ala Pro Arg Arg Gly Gln LysThr Arg Gln Lys 115 120 125 Asn Thr Ser Ala His Phe Leu Pro Met Val ValHis Ser 130 135 140 32 base pairs nucleic acid single linear cDNA 125GCGGCACATG TCTTACAACC ACCTGCAGGG TG 32 84 base pairs nucleic acid singlelinear cDNA 126 CTGCCCAAGC TTTTATGAGT GTACCACCAT TGGAAGAAAG TGAGCAGAGGTGTTTTTCCT 60 TCGTGTTTCC TGTCCTCTCC TTGG 84 426 base pairs nucleic aciddouble linear cDNA 127 ATGTCTTACA ACCACCTGCA GGGTGACGTT CGTTGGCGTAAACTGTTCTC TTTCACCAAA 60 TACTTCCTGA AAATCGAAAA AAACGGTAAA GTTTCTGGGACCAAGAAGGA GAACTGCCCG 120 TACAGCATCC TGGAGATAAC ATCAGTAGAA ATCGGAGTTGTTGCCGTCAA AGCCATTAAC 180 AGCAACTATT ACTTAGCCAT GAACAAGAAG GGGAAACTCTATGGCTCAAA AGAATTTAAC 240 AATGACTGTA AGCTGAAGGA GAGGATAGAG GAAAATGGATACAATACCTA TGCATCATTT 300 AACTGGCAGC ATAATGGGAG GCAAATGTAT GTGGCATTGAATGGAAAAGG AGCTCCAAGG 360 AGAGGACAGG AAACACGAAG GAAAAACACC TCTGCTCACTTTCTTCCAAT GGTGGTACAC 420 TCATAG 426 141 amino acids amino acid singleNot Relevant protein 128 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg TrpArg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu LysAsn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser IleLeu Glu Ile Thr Ser 35 40 45 Val Glu Ile Gly Val Val Ala Val Lys Ala IleAsn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr GlySer Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg Ile GluGlu Asn Gly Tyr Asn Thr 85 90 95 Tyr Ala Ser Phe Asn Trp Gln His Asn GlyArg Gln Met Tyr Val Ala 100 105 110 Leu Asn Gly Lys Gly Ala Pro Arg ArgGly Gln Glu Thr Arg Arg Lys 115 120 125 Asn Thr Ser Ala His Phe Leu ProMet Val Val His Ser 130 135 140 32 base pairs nucleic acid single linearcDNA 129 GCGGCACATG TCTTACAACC ACCTGCAGGG TG 32 84 base pairs nucleicacid single linear cDNA 130 CTGCCCAAGC TTTTATGAGT GTACCACCAT TGGAAGAAAGTGAGCAGAGG TGTTTTTCCT 60 TCGTGTCTGC TGTCCTCTCC TTGG 84 426 base pairsnucleic acid double linear cDNA 131 ATGTCTTACA ACCACCTGCA GGGTGACGTTCGTTGGCGTA AACTGTTCTC TTTCACCAAA 60 TACTTCCTGA AAATCGAAAA AAACGGTAAAGTTTCTGGGA CCAAGAAGGA GAACTGCCCG 120 TACAGCATCC TGGAGATAAC ATCAGTAGAAATCGGAGTTG TTGCCGTCAA AGCCATTAAC 180 AGCAACTATT ACTTAGCCAT GAACAAGAAGGGGAAACTCT ATGGCTCAAA AGAATTTAAC 240 AATGACTGTA AGCTGAAGGA GAGGATAGAGGAAAATGGAT ACAATACCTA TGCATCATTT 300 AACTGGCAGC ATAATGGGAG GCAAATGTATGTGGCATTGA ATGGAAAAGG AGCTCCAAGG 360 AGAGGACAGC AGACACGAAG GAAAAACACCTCTGCTCACT TTCTTCCAAT GGTGGTACAC 420 TCATAG 426 141 amino acids aminoacid single Not Relevant protein 132 Met Ser Tyr Asn His Leu Gln Gly AspVal Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu LysIle Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys ProTyr Ser Ile Leu Glu Ile Thr Ser 35 40 45 Val Glu Ile Gly Val Val Ala ValLys Ala Ile Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly LysLeu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys GluArg Ile Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Ala Ser Phe Asn Trp GlnHis Asn Gly Arg Gln Met Tyr Val Ala 100 105 110 Leu Asn Gly Lys Gly AlaPro Arg Arg Gly Gln Gln Thr Arg Arg Lys 115 120 125 Asn Thr Ser Ala HisPhe Leu Pro Met Val Val His Ser 130 135 140 32 base pairs nucleic acidsingle linear cDNA 133 GCGGCACATG TCTTACAACC ACCTGCAGGG TG 32 93 basepairs nucleic acid single linear cDNA 134 CTGCCCAAGC TTTTATGAGTGTACCACCAT TGGAAGAAAG TGAGCAGAGG TGTTTTTCCT 60 TCGTGTTTTC TGTCCTTCCCTTGGAGCTCC TTT 93 426 base pairs nucleic acid double linear cDNA 135ATGTCTTACA ACCACCTGCA GGGTGACGTT CGTTGGCGTA AACTGTTCTC TTTCACCAAA 60TACTTCCTGA AAATCGAAAA AAACGGTAAA GTTTCTGGGA CCAAGAAGGA GAACTGCCCG 120TACAGCATCC TGGAGATAAC ATCAGTAGAA ATCGGAGTTG TTGCCGTCAA AGCCATTAAC 180AGCAACTATT ACTTAGCCAT GAACAAGAAG GGGAAACTCT ATGGCTCAAA AGAATTTAAC 240AATGACTGTA AGCTGAAGGA GAGGATAGAG GAAAATGGAT ACAATACCTA TGCATCATTT 300AACTGGCAGC ATAATGGGAG GCAAATGTAT GTGGCATTGA ATGGAAAAGG AGCTCCAAGG 360GAAGGACAGA AAACACGAAG GAAAAACACC TCTGCTCACT TTCTTCCAAT GGTGGTACAC 420TCATAG 426 140 amino acids amino acid single Not Relevant protein 136Met Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe Ser 1 5 1015 Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys Val Ser Gly 20 2530 Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr Ser Val 35 4045 Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr Leu 50 5560 Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn 65 7075 80 Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr Tyr 8590 95 Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala Leu100 105 110 Asn Gly Lys Gly Ala Pro Arg Glu Gly Gln Lys Thr Arg Arg LysAsn 115 120 125 Thr Ser Ala His Phe Leu Pro Met Val Val His Ser 130 135140 32 base pairs nucleic acid single linear cDNA 137 GCGGCACATGTCTTACAACC ACCTGCAGGG TG 32 93 base pairs nucleic acid single linearcDNA 138 CTGCCCAAGC TTTTATGAGT GTACCACCAT TGGAAGAAAG TGAGCAGAGGTGTTTTTCCT 60 TCGTGTTTTC TGTCCCTGCC TTGGAGCTCC TTT 93 426 base pairsnucleic acid double linear cDNA 139 ATGTCTTACA ACCACCTGCA GGGTGACGTTCGTTGGCGTA AACTGTTCTC TTTCACCAAA 60 TACTTCCTGA AAATCGAAAA AAACGGTAAAGTTTCTGGGA CCAAGAAGGA GAACTGCCCG 120 TACAGCATCC TGGAGATAAC ATCAGTAGAAATCGGAGTTG TTGCCGTCAA AGCCATTAAC 180 AGCAACTATT ACTTAGCCAT GAACAAGAAGGGGAAACTCT ATGGCTCAAA AGAATTTAAC 240 AATGACTGTA AGCTGAAGGA GAGGATAGAGGAAAATGGAT ACAATACCTA TGCATCATTT 300 AACTGGCAGC ATAATGGGAG GCAAATGTATGTGGCATTGA ATGGAAAAGG AGCTCCAAGG 360 CAGGGACAGA AAACACGAAG GAAAAACACCTCTGCTCACT TTCTTCCAAT GGTGGTACAC 420 TCATAG 426 141 amino acids aminoacid single Not Relevant protein 140 Met Ser Tyr Asn His Leu Gln Gly AspVal Arg Trp Arg Lys Leu Phe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu LysIle Glu Lys Asn Gly Lys Val Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys ProTyr Ser Ile Leu Glu Ile Thr Ser 35 40 45 Val Glu Ile Gly Val Val Ala ValLys Ala Ile Asn Ser Asn Tyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly LysLeu Tyr Gly Ser Lys Glu Phe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys GluArg Ile Glu Glu Asn Gly Tyr Asn Thr 85 90 95 Tyr Ala Ser Phe Asn Trp GlnHis Asn Gly Arg Gln Met Tyr Val Ala 100 105 110 Leu Asn Gly Lys Gly AlaPro Arg Gln Gly Gln Lys Thr Arg Arg Lys 115 120 125 Asn Thr Ser Ala HisPhe Leu Pro Met Val Val His Ser 130 135 140 32 base pairs nucleic acidsingle linear cDNA 141 GCGGCACATG TCTTACAACC ACCTGCAGGG TG 32 21 basepairs nucleic acid single linear cDNA 142 TTGAATGGAG AAGGAGCTCC A 21 21base pairs nucleic acid single linear cDNA 143 TGGAGCTCCT TCTCCATTCA A21 33 base pairs nucleic acid single linear cDNA 144 CTGCCCAAGCTTTTATGAGT GTACCACCAT TGG 33 426 base pairs nucleic acid double linearcDNA 145 ATGTCTTACA ACCACCTGCA GGGTGACGTT CGTTGGCGTA AACTGTTCTCTTTCACCAAA 60 TACTTCCTGA AAATCGAAAA AAACGGTAAA GTTTCTGGGA CCAAGAAGGAGAACTGCCCG 120 TACAGCATCC TGGAGATAAC ATCAGTAGAA ATCGGAGTTG TTGCCGTCAAAGCCATTAAC 180 AGCAACTATT ACTTAGCCAT GAACAAGAAG GGGAAACTCT ATGGCTCAAAAGAATTTAAC 240 AATGACTGTA AGCTGAAGGA GAGGATAGAG GAAAATGGAT ACAATACCTATGCATCATTT 300 AACTGGCAGC ATAATGGGAG GCAAATGTAT GTGGCATTGA ATGGAGAAGGAGCTCCAAGG 360 AGAGGACAGA AAACACGAAG GAAAAACACC TCTGCTCACT TTCTTCCAATGGTGGTACAC 420 TCATAG 426 141 amino acids amino acid single Not Relevantprotein 146 Met Ser Tyr Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys LeuPhe 1 5 10 15 Ser Phe Thr Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly LysVal Ser 20 25 30 Gly Thr Lys Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu IleThr Ser 35 40 45 Val Glu Ile Gly Val Val Ala Val Lys Ala Ile Asn Ser AsnTyr Tyr 50 55 60 Leu Ala Met Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys GluPhe Asn 65 70 75 80 Asn Asp Cys Lys Leu Lys Glu Arg Ile Glu Glu Asn GlyTyr Asn Thr 85 90 95 Tyr Ala Ser Phe Asn Trp Gln His Asn Gly Arg Gln MetTyr Val Ala 100 105 110 Leu Asn Gly Glu Gly Ala Pro Arg Arg Gly Gln LysThr Arg Arg Lys 115 120 125 Asn Thr Ser Ala His Phe Leu Pro Met Val ValHis Ser 130 135 140 3974 base pairs nucleic acid both both cDNA 147GGTACCTAAG TGAGTAGGGC GTCCGATCGA CGGACGCCTT TTTTTTGAAT TCGTAATCAT 60GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG 120CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG 180CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA 240TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA 300CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG 360TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC 420AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC 480CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC 540TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC 600TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA 660GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC 720ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA 780ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG 840CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA 900GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG 960GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC 1020AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT 1080CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCGTCGA 1140CAATTCGCGC GCGAAGGCGA AGCGGCATGC ATTTACGTTG ACACCATCGA ATGGTGCAAA 1200ACCTTTCGCG GTATGGCATG ATAGCGCCCG GAAGAGAGTC AATTCAGGGT GGTGAATGTG 1260AAACCAGTAA CGTTATACGA TGTCGCAGAG TATGCCGGTG TCTCTTATCA GACCGTTTCC 1320CGCGTGGTGA ACCAGGCCAG CCACGTTTCT GCGAAAACGC GGGAAAAAGT GGAAGCGGCG 1380ATGGCGGAGC TGAATTACAT TCCCAACCGC GTGGCACAAC AACTGGCGGG CAAACAGTCG 1440TTGCTGATTG GCGTTGCCAC CTCCAGTCTG GCCCTGCACG CGCCGTCGCA AATTGTCGCG 1500GCGATTAAAT CTCGCGCCGA TCAACTGGGT GCCAGCGTGG TGGTGTCGAT GGTAGAACGA 1560AGCGGCGTCG AAGCCTGTAA AGCGGCGGTG CACAATCTTC TCGCGCAACG CGTCAGTGGG 1620CTGATCATTA ACTATCCGCT GGATGACCAG GATGCCATTG CTGTGGAAGC TGCCTGCACT 1680AATGTTCCGG CGTTATTTCT TGATGTCTCT GACCAGACAC CCATCAACAG TATTATTTTC 1740TCCCATGAAG ACGGTACGCG ACTGGGCGTG GAGCATCTGG TCGCATTGGG TCACCAGCAA 1800ATCGCGCTGT TAGCGGGCCC ATTAAGTTCT GTCTCGGCGC GTCTGCGTCT GGCTGGCTGG 1860CATAAATATC TCACTCGCAA TCAAATTCAG CCGATAGCGG AACGGGAAGG CGACTGGAGT 1920GCCATGTCCG GTTTTCAACA AACCATGCAA ATGCTGAATG AGGGCATCGT TCCCACTGCG 1980ATGCTGGTTG CCAACGATCA GATGGCGCTG GGCGCAATGC GCGCCATTAC CGAGTCCGGG 2040CTGCGCGTTG GTGCGGATAT CTCGGTAGTG GGATACGACG ATACCGAAGA CAGCTCATGT 2100TATATCCCGC CGTTAACCAC CATCAAACAG GATTTTCGCC TGCTGGGGCA AACCAGCGTG 2160GACCGCTTGC TGCAACTCTC TCAGGGCCAG GCGGTGAAGG GCAATCAGCT GTTGCCCGTC 2220TCACTGGTGA AAAGAAAAAC CACCCTGGCG CCCAATACGC AAACCGCCTC TCCCCGCGCG 2280TTGGCCGATT CATTAATGCA GCTGGCACGA CAGGTTTCCC GACTGGAAAG CGGGCAGTGA 2340GCGCAACGCA ATTAATGTAA GTTAGCGCGA ATTGTCGACC AAAGCGGCCA TCGTGCCTCC 2400CCACTCCTGC AGTTCGGGGG CATGGATGCG CGGATAGCCG CTGCTGGTTT CCTGGATGCC 2460GACGGATTTG CACTGCCGGT AGAACTCCGC GAGGTCGTCC AGCCTCAGGC AGCAGCTGAA 2520CCAACTCGCG AGGGGATCGA GCCCGGGGTG GGCGAAGAAC TCCAGCATGA GATCCCCGCG 2580CTGGAGGATC ATCCAGCCGG CGTCCCGGAA AACGATTCCG AAGCCCAACC TTTCATAGAA 2640GGCGGCGGTG GAATCGAAAT CTCGTGATGG CAGGTTGGGC GTCGCTTGGT CGGTCATTTC 2700GAACCCCAGA GTCCCGCTCA GAAGAACTCG TCAAGAAGGC GATAGAAGGC GATGCGCTGC 2760GAATCGGGAG CGGCGATACC GTAAAGCACG AGGAAGCGGT CAGCCCATTC GCCGCCAAGC 2820TCTTCAGCAA TATCACGGGT AGCCAACGCT ATGTCCTGAT AGCGGTCCGC CACACCCAGC 2880CGGCCACAGT CGATGAATCC AGAAAAGCGG CCATTTTCCA CCATGATATT CGGCAAGCAG 2940GCATCGCCAT GGGTCACGAC GAGATCCTCG CCGTCGGGCA TGCGCGCCTT GAGCCTGGCG 3000AACAGTTCGG CTGGCGCGAG CCCCTGATGC TCTTCGTCCA GATCATCCTG ATCGACAAGA 3060CCGGCTTCCA TCCGAGTACG TGCTCGCTCG ATGCGATGTT TCGCTTGGTG GTCGAATGGG 3120CAGGTAGCCG GATCAAGCGT ATGCAGCCGC CGCATTGCAT CAGCCATGAT GGATACTTTC 3180TCGGCAGGAG CAAGGTGAGA TGACAGGAGA TCCTGCCCCG GCACTTCGCC CAATAGCAGC 3240CAGTCCCTTC CCGCTTCAGT GACAACGTCG AGCACAGCTG CGCAAGGAAC GCCCGTCGTG 3300GCCAGCCACG ATAGCCGCGC TGCCTCGTCC TGCAGTTCAT TCAGGGCACC GGACAGGTCG 3360GTCTTGACAA AAAGAACCGG GCGCCCCTGC GCTGACAGCC GGAACACGGC GGCATCAGAG 3420CAGCCGATTG TCTGTTGTGC CCAGTCATAG CCGAATAGCC TCTCCACCCA AGCGGCCGGA 3480GAACCTGCGT GCAATCCATC TTGTTCAATC ATGCGAAACG ATCCTCATCC TGTCTCTTGA 3540TCAGATCTTG ATCCCCTGCG CCATCAGATC CTTGGCGGCA AGAAAGCCAT CCAGTTTACT 3600TTGCAGGGCT TCCCAACCTT ACCAGAGGGC GCCCCAGCTG GCAATTCCGG TTCGCTTGCT 3660GTCCATAAAA CCGCCCAGTC TAGCTATCGC CATGTAAGCC CACTGCAAGC TACCTGCTTT 3720CTCTTTGCGC TTGCGTTTTC CCTTGTCCAG ATAGCCCAGT AGCTGACATT CATCCGGGGT 3780CAGCACCGTT TCTGCGGACT GGCTTTCTAC GTGTTCCGCT TCCTTTAGCA GCCCTTGCGC 3840CCTGAGTGCT TGCGGCAGCG TGAAGCTTAA AAAACTGCAA AAAATAGTTT GACTTGTGAG 3900CGGATAACAA TTAAGATGTA CCCAATTGTG AGCGGATAAC AATTTCACAC ATTAAAGAGG 3960AGAAATTACA TATG 3974 112 base pairs nucleic acid both linear cDNA 148AAGCTTAAAA AACTGCAAAA AATAGTTTGA CTTGTGAGCG GATAACAATT AAGATGTACC 60CAATTGTGAG CGGATAACAA TTTCACACAT TAAAGAGGAG AAATTACATA TG 112

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence at least 95% identical to Gly (138)-Ser (208) of SEQ ID NO: 2.2. The isolated polypeptide of claim 1, wherein said amino acid sequenceis at least 95% identical to Val (123)-Ser (208) of SEQ ID NO:
 2. 3. Theisolated polypeptide of claim 2, wherein said amino acid sequence is atleast 95% identical to Glu (104)-Ser (208) of SEQ ID NO:
 2. 4. theisolated polypeptide of claim 3, wherein said amino acid sequence is atleast 95% identical to Val (77)-Ser (208) of SEQ ID NO:
 2. 5. Theisolated polypeptide of claim 4, wherein said amino acid sequence is atleast 95% identical to Ser (69)-Ser (208) of SEQ ID NO:
 2. 6. Theisolated polypeptide of claim 5, wherein said amino acid sequence is atleast 95% identical to Ala (63)-Ser (208) of SEQ ID NO:
 2. 7. Theisolated polypeptide of claim 6, wherein said amino acid *sequence is atleast 95% identical to Cys (37)-Ser (208) of SEQ ID NO:
 2. 8. Theisolated polypeptide of claim 7, wherein said amino acid sequence is atleast 95% identical to Thr (36)-Ser (208) of SEQ ID NO:
 2. 9. Theisolated polypeptide of claim 8, wherein said amino acid sequence is atleast 95% identical to Trp (2)-Ser (208) of SEQ ID NO:
 2. 10. Theisolated polypeptide of claim 9, wherein said amino acid sequence is atleast 95% identical to Met (1)-Ser (208) of SEQ ID NO:
 2. 11. Anisolated polypeptide comprising an amino acid sequence at least 95%identical to Ala (63)-Lys (153) of SEQ ID NO:
 2. 12. The isolatedpolypeptide of claim 11, wherein said amino acid sequence is at least95% identical to Thr (36)-Lys (153) of SEQ ID NO:
 2. 13. An isolatedpolypeptide comprising Gly (138)-Ser (208) of SEQ ID NO:
 2. 14. Theisolated polypeptide of claim 13, which comprises Val (123)-Ser (208) ofSEQ ID NO:
 2. 15. The isolated polypeptide of claim 14, which comprisesGlu (104)-Ser (208) of SEQ ID NO:
 2. 16. The isolated polypeptide ofclaim 15, which comprises Val (77)-Ser (208) of SEQ ID NO:
 2. 17. Theisolated polypeptide of claim 16, which comprises Ser (69)-Ser (208) ofSEQ ID NO:
 2. 18. The isolated polypeptide of claim 17, which comprisesAla (63)-Ser (208) of SEQ ID NO:
 2. 19. The isolated polypeptide ofclaim 18, which comprises Cys (37)-Ser (208) of SEQ ID NO:
 2. 20. Theisolated polypeptide of claim 19, which comprises Thr (36)-Ser (208) ofSEQ ID NO:
 2. 21. The isolated polypeptide of claim 20, which comprisesTrp (2)-Ser (208) of SEQ ID NO:
 2. 22. The isolated polypeptide of claim21, which comprises Met (1)-Ser (208) of SEQ ID NO:
 2. 23. An isolatedpolypeptide comprising Ala (63)-Lys (153) of SEQ ID NO:
 2. 24. Theisolated polypeptide of claim 23, which comprises Thr (36)-Lys (153) ofSEQ ID NO:
 2. 25. An isolated polypeptide consisting essentially of anamino acid sequence at least 95% identical to Ser (69)-Ser (208) of SEQID NO:
 2. 26. The isolated polypeptide of claim 25, wherein said aminoacid sequence is at least 97% identical to Ser (69)-Ser (208) of SEQ IDNO:
 2. 27. The isolated polypeptide of claim 26, wherein said amino acidsequence is at least 99% identical to Ser (69)-Ser (208) of SEQ ID NO:2.
 28. An isolated polypeptide consisting essentially of Ser (69)-Ser(208) of SEQ ID NO:
 2. 29. The isolated polypeptide of any one of claims1, 5, 11, 13, 17, 23, or 25, having a Met residue at the N-terminus. 30.The isolated polypeptide of claim 28, having a Met residue at theN-terminus.
 31. The isolated polypeptide of claim 1 or 13, wherein saidamino acid sequence includes one or more amino acid substitutionsselected from Gly (142) Ala, Ser (143) Lys, Phe (146) Ser, Asn (148)Glu, Lys (151) Asn, Leu (152) Phe, Glu (154) Gly, Glu (154) Asp, Arg(155) Leu, Glu (157) Leu, Gly (160) His, Phe (167) Ala, Asn (168) Lys,Gln (170) Thr, Arg (174) Gly, Tyr (177) Phe, Gly (182) Gln, Ala (185)Val, Ala (185) Leu, Ala (185) Ile, Arg (187) Gln (190) Lys, Lys (195)Glu, Thr (197) Lys, or Ser (198) Thr.
 32. The isolated polypeptide ofclaim 5, 17, 25, or 28 wherein said amino acid sequence includes one ormore amino acid substitutions selected from Trp (79) Val, Arg (80) Lys,Lys (87) Arg, Tyr (88) Trp, Phe (89) Tyr, Lys (91) Arg, Ser (99) Lys,Lys (102) Gln, Lys 103(Glu), Glu (104) Met, Asn (105) Lys, Pro (107)Asn, Ser (109) Asn, Leu (111) Met, Thr (114) Arg, Glu(117) Ala, Val(120) Ile, Val (123) Ile, Ala (125) Gly, Ile (126) Val, Asn (127) Glu,Asn (127) Gln, Tyr (130) Phe, Met (134) Thr, Lys (136) Glu, Lys (137)Glu, Gly (142) Ala, Ser (143) Lys, Phe (146) Ser, Asn (148) Glu, Lys(151) Asn, Leu (52) Phe, Glu (154) Gly, Glu (154) Asp, Arg (155) Leu,Glu (157) Leu, Gly (160) His, Phe (167) Ala, Asn (168) Lys, Gln (170)Thr, Arg (174) Gly, Tyr (177) Phe, Gly (182) Gln, Ala (185) Val, Ala(185) Leu, Ala (185) Ile, Arg (187) Gln (190) Lys, Lys (195) Glu, Thr(197) Lys, Ser (198) Thr, Arg (194) Glu, Arg (194) Gln, Lys (191) Glu,Lys (191) Gln, Arg (188) Glu, Arg (188) Gln, or Lys (183) Glu.
 33. Theisolated polypeptide of claim 11 or 23, wherein said amino acid sequenceincludes one or more amino acid substitutions selected from Ala (63)Pro, Gly (64) Glu, Val (67) Thr, Trp (79) Val, Arg (80) Lys, Lys (87)Arg, Tyr (88) Trp, Phe (89) Tyr, Lys (91) Arg, Ser (99) Lys, Lys (102)Gln, Lys 103(Glu), Glu (104) Met, Asn (105) Lys, Pro (107) Asn, Ser(109) Asn, Leu (111) Met, Thr (114) Arg, Glu(117) Ala, Val (120) Ile,Val (123) Ile, Ala (125) Gly, Ile (126) Val, Asn (127) Glu, Asn (127)Gln, Tyr (130) Phe, Met (134) Thr, Lys (136) Glu, Lys (137) Glu, Gly(142) Ala, Ser (143) Lys, Phe (146) Ser, Asn (148) Glu, Lys (151) Asn,or Leu (152) Phe.
 34. The isolated polypeptide of any one of claims 1,5, 11, 13, 17, 23, 25, or 28, which is part of a fusion protein.
 35. Theisolated polypeptide of claim 34, wherein said polypeptide is fused to amarker.
 36. The isolated polypeptide of claim 35, wherein said marker isselected from the group consisting of a hexahistidine tag and ahemagglutinin tag.
 37. The isolated polypeptide of any one of claims 1,5, 11, 13, 17, 23, 25, or 28, which stimulates proliferation ofepithelial cells.
 38. The isolated polypeptide of any one of claims 1,5, 11, 13, 17, 23, 25,or 28,which stimulates proliferation ofkeratinocytes.
 39. The isolated polypeptide of any one of claims 1, 5,11, 13, 17, 23, 25,or 28,which is produced in a recombinant host cell.40. The isolated polypeptide of claim 39, wherein said host cell ismammalian.
 41. The isolated polypeptide of claim 39, wherein said hostcell is bacterial.
 42. The isolated polypeptide of claim 41, whereinsaid host cell is E. coli.
 43. An isolated polypeptide comprising ahydrophilic region of Keratinocyte Growth Factor-2 (KGF-2) selected fromthe group consisting of Gly (41)-Asn (71), Lys (91)-Ser (109), Asn(135)-Tyr (164), and Asn (181)-Ala (199) of SEQ ID NO:
 2. 44. Thepolypeptide of claim 43, which is not more than 100 amino acids inlength.
 45. The polypeptide of claim 44, which is not more than 50 aminoacids in length.
 46. The isolated polypeptide of any one of claims 1, 5,11, 13, 17, 23, or 25,together with a pharmaceutically acceptablecarrier or excipient.
 47. The isolated polypeptide of claim 28, togetherwith a pharmaceutically acceptable carrier or excipient.
 48. An isolatedpolynucleotide encoding a polypeptide of any one of claims 1, 5, 11, 13,17, 23, 25, or
 28. 49. The isolated polynucleotide of claim 48, which isoptimized for expression in E. coli.
 50. The isolated polynucleotide ofclaim 49, having the nucleotide sequence of SEQ ID NO:
 38. 51. Theisolated polynucleotide of claim 49, having the nucleotide sequence ofSEQ ID NO:
 42. 52. The isolated polynucleotide of claim 49, having thenucleotide sequence of SEQ ID NO:
 54. 53. The isolated polynucleotide ofclaim 49, having the nucleotide sequence of SEQ ID NO:
 111. 54. A methodfor making a recombinant vector comprising inserting the polynulceotideof claim 48 into a vector.
 55. A recombinant vector produced by themethod of claim
 54. 56. A method of making a recombinant host cellcomprising introducing the recombinant vector of claim 55 into a hostcell.
 57. A recombinant host cell produced by the method of claim 56.58. The isolated polypeptide of claim 1, 5, 11, 13, 17, 23, 25,or 28,which is produced by a method comprising: introducing a recombinantvector comprising a polynucleotide encoding said polypeptide into a hostcell; culturing said host cell; and recovering said polypeptide.
 59. Amethod for producing a polypeptide comprising: culturing the recombinanthost cell of claim 57 under conditions that said vector is expressed;and recovering said polypeptide.
 60. A method of stimulatingproliferation of epidermal cells comprising administering an effectiveamount of the polypeptide of claim
 1. 61. The metod of claim 60, whereinthe epidermal cells are keratinocytes.
 62. The method of claim 61,wherein said polypeptide is administered to an individual.
 63. Themethod of claim 62, wherein said polypeptide is administered for apurpose selected from: preventing or improving the appearance ofwrinkles or aged skin, improving skin strength, promoting epidermalthickening, reducing scarring, or improving healing after cosmeticsurgery.
 64. A method of stimulating proliferation of epithelial cellscomprising administering an effective amount of the polypeptide ofclaim
 1. 65. The method of claim 64, wherein said epithelial cells areselected from epithelial cells of the liver, pancreas, kidney, prostate,bladder, lung and esophagus.
 66. A method of promoting wound healingcomprising administering an effective amount of the polypeptide of claim1 to an individual in need thereof.
 67. The method of claim 66, whereinsaid individual is wound healing impaired.
 68. The method of claim 67,wherein said impairment in wound healing is caused by diabetes, ischemicblockage or injury, steriods, non-steroid compounds, uremia,malnutrition, vitamin deficiencies, obesity, infection,immunosuppression, radiation therapy, or chemotherapy.
 69. The method ofclaim 66, wherein said wound is selected from surgical wounds,excisional wounds, deep wounds involving damage of the dermis andepidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds,diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venousstasis ulcers, or burns.
 70. A method of treating wounds caused by acolonic or gastrointestinal (GI) surgical procedure comprisingadministering an effective amount of the polypeptide of claim 1 to anindividual in need thereof.
 71. The method of claim 70, wherein saidprocedure is anastomosis.
 72. The method of claim 70, wherein saidindividual is wound healing impaired.
 73. The method of claim 72,wherein said impairment is caused by diabetes, ischemic blockage orinjury, steroids, non-steroid compounds, uremia, malnutrition, vitamindeficiencies, obesity, infection, immuno suppression, radiation therapy,or chemotherapy.
 74. A method of treating or preventing mucositiscomprising administering an effective amount of the polypeptide of claim1 to an individual in need thereof.
 75. The method of claim 74, whereinsaid mucositis is selected from oral, esophageal, gastric, intestinal,colonic, rectal or anal.
 76. A method of treating inflammatory boweldisease comprising administering an effective amount of the polypeptideof claim 1 to an individual in need thereof.
 77. The method of claim 76wherein said disease is selected from ulcerative colitis or Crohn'sdisease.
 78. A method of reducing inflammation comprising administeringan effective amount of the polypeptide of claim 1 to an individual inneed thereof.
 79. The method of claim 78, wherein said inflammation isassociated with a disease or condition selected from psoriasis, eczema,dermatitis, or arthritis.
 80. A method of promoting hair growthcomprising administering an effective amount of the polypeptide of claim1 to an individual in need thereof.
 81. A method of treating tissueexposed to radiation or protecting tissue to be exposed to radiationcomprising administering an effective amount of the polypeptide of claim1 to an individual in need thereof.
 82. The method of claim 81, whereinsaid polypeptide is administered to allow an increase in radiationdosage used to treat a malignancy in said individual.
 83. The method ofclaim 82, wherein said polypeptide is administered to treat aradiation-induced condition selected from oral injury, gastro-intestinalinjury, mucositis, intestinal fibrosis, proctitis, pulmonary fibrosis,pneumonitis, pleural retraction, hemopoietic syndrome, or myelotoxicity.84. A method of promoting urothelial healing comprising administering aneffective amount of the polypeptide of claim 1 to an individual in needthereof.
 85. A method of promoting tissue growth or repair in the femalegenital tract comprising administering an effective amount of thepolypeptide of claim 1 to an individual in need thereof.
 86. A method oftreating or preventing viral hepatitis comprising administering aneffective amount of the polypeptide of claim 1 to an individual in needthereof.
 87. A method of treating or preventing liver failure comprisingadministering an effective amount of the polypeptide of claim 1 to anindividual in need thereof.
 88. The method of claim 87, wherein theliver failure is caused by a disease or condition selected from acuteviral hepatitis, cirrhosis, drug-induced hepatitis, toxin-inducedhepatitis, autoimmune chronic active hepatitis, liver transplantation,or partial hepatectomy.
 89. The method of claim 88, wherein saidtoxin-induced hepatitis is caused by a hepatotoxin selected fromacetaminophen, carbon tetrachloride, methotrexate, or an organicarsenical.
 90. A method of treating or preventing pancreatitiscomprising administering an effective amount of the polypeptide of claim1 to an individual in need thereof.
 91. A method of treating orpreventing a lung damaging condition comprising administering aneffective amount of the polypeptide of claim 1 to an individual in needthereof.
 92. The method of claim 91, wherein the condition is selectedfrom emphysema, inhalation injury, chemotherapy, radiation treatment,lung cancer, asthma, respiratory distress syndrome, or bronchopulmonarydysplasia.
 93. A method of treating or preventing renal failurecomprising administering an effective amount of the polypeptide of claim1 to an individual in need thereof.
 94. An isolated polypeptideconsisting of Met (1)-Ser (141) in SEQ ID NO: 96.